LOCUS Exported 10685 bp ds-DNA circular SYN 19-DEC-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..10685
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 532..1120
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 1306..1445
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(1547..1738)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(2547..3629)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=lacI
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(3630..3707)
/gene="lacI"
/label=lacI promoter
promoter 4016..4034
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4035..4059
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 4074..4096
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4110..4127
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
misc_feature 4128..5243
/label=MBP
CDS 5244..5261
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 5268..5288
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
misc_feature 5289..9188
/label=FnCpf1 (humanized)
CDS 9189..9236
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 9243..9323
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
terminator 9516..9563
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 9600..10055
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10081..10185
/gene="bla"
/label=AmpR promoter
CDS 10186..361
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
ORIGIN
1 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
61 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
181 gtttattgct gataaatctg gagccggtga gcgtggtagt cgcggtatca ttgcagcact
241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
661 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
1021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
1081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
1141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
1201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
1261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
1321 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
1381 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
1441 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
1501 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
1561 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
1621 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
1681 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
1741 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
1801 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
1861 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
1921 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
1981 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
2041 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
2101 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
2161 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
2221 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
2281 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
2341 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
2401 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
2461 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
2521 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
2581 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
2641 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
2701 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
2761 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
2821 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
2881 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
2941 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
3001 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
3061 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
3121 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
3181 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
3241 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
3301 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
3361 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
3421 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
3481 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
3541 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
3601 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
3661 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
3721 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
3781 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
3841 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
3901 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
3961 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
4021 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
4081 aactttaaga aggagatata catatgggtc atcatcatca ccatcacaaa atcgaagaag
4141 gcaaactggt tatttggatc aatggcgata aaggctataa tggtctggca gaagttggca
4201 aaaaattcga aaaagatacc ggcattaaag tgaccgttga acatccggat aaactggaag
4261 aaaaatttcc gcaggttgca gcaaccggtg atggtccgga tattatcttt tgggcacatg
4321 atcgttttgg tggttatgca cagagcggtc tgctggcaga aattacaccg gataaagcat
4381 ttcaggacaa actgtatccg tttacctggg atgcagttcg ctataacggt aaactgattg
4441 catatccgat tgcagttgaa gcactgagcc tgatctataa caaagatctg ctgccgaatc
4501 cgcctaaaac ctgggaagaa attccggcac tggataaaga actgaaagca aaaggtaaaa
4561 gcgcactgat gtttaatctg caagaaccgt attttacctg gcctctgatt gcagcagatg
4621 gtggctatgc attcaaatat gaaaacggca aatatgatat caaagacgtg ggtgttgata
4681 atgcgggtgc aaaagccggt ctgacctttc tggttgatct gattaaaaac aaacacatga
4741 acgccgatac cgattatagc attgcagaag cagcatttaa caaaggtgaa accgcaatga
4801 caattaatgg tccgtgggca tggtcaaata ttgataccag caaagtgaat tatggtgtta
4861 ccgttctgcc gacatttaaa ggtcagccga gcaaaccgtt tgttggtgtt ctgagcgcag
4921 gtattaatgc agcaagcccg aacaaagaac tggcaaaaga atttctggaa aactatctgc
4981 tgaccgatga aggtctggaa gcagtgaata aagataaacc gctgggtgca gttgcactga
5041 aaagctatga agaagaactg gttaaagatc cgcgtattgc agccaccatg gaaaatgcac
5101 agaaaggcga aattatgccg aatattccgc agatgagcgc attttggtat gccgttcgta
5161 ccgcagtgat taatgccgca tcaggtcgtc agaccgttga tgaagccctg aaagatgccc
5221 agaccggtgc aagcaccgga tccctggttc cgcgtggtag cgctagcgaa aatctgtatt
5281 ttcagggaat gagcatctac caggagttcg tcaacaagta ttcactgagt aagacactgc
5341 ggttcgagct gatcccacag ggcaagacac tggagaacat caaggcccga ggcctgattc
5401 tggacgatga gaagcgggca aaagactata agaaagccaa gcagatcatt gataaatacc
5461 accagttctt tatcgaggaa attctgagct ccgtgtgcat cagtgaggat ctgctgcaga
5521 attactcaga cgtgtacttc aagctgaaga agagcgacga tgacaacctg cagaaggact
5581 tcaagtccgc caaggacacc atcaagaaac agattagcga gtacatcaag gactccgaaa
5641 agtttaaaaa tctgttcaac cagaatctga tcgatgctaa gaaaggccag gagtccgacc
5701 tgatcctgtg gctgaaacag tctaaggaca atgggattga actgttcaag gctaactccg
5761 atatcactga tattgacgag gcactggaaa tcatcaagag cttcaaggga tggaccacat
5821 actttaaagg cttccacgag aaccgcaaga acgtgtactc cagcaacgac attcctacct
5881 ccatcatcta ccgaatcgtc gatgacaatc tgccaaagtt cctggagaac aaggccaaat
5941 atgaatctct gaaggacaaa gctcccgagg caattaatta cgaacagatc aagaaagatc
6001 tggctgagga actgacattc gatatcgact ataagactag cgaggtgaac cagagggtct
6061 tttccctgga cgaggtgttt gaaatcgcca atttcaacaa ttacctgaac cagtccggca
6121 ttactaaatt caataccatc attggcggga agtttgtgaa cggggagaat accaagcgca
6181 agggaattaa cgaatacatc aatctgtata gccagcagat caacgacaaa actctgaaga
6241 aatacaagat gtctgtgctg ttcaaacaga tcctgagtga taccgagtcc aagtcttttg
6301 tcattgataa actggaagat gactcagacg tggtcactac catgcagagc ttttatgagc
6361 agatcgccgc tttcaagaca gtggaggaaa aatctattaa ggaaactctg agtctgctgt
6421 tcgatgacct gaaagcccag aagctggacc tgagtaagat ctacttcaaa aacgataaga
6481 gtctgacaga cctgtcacag caggtgtttg atgactattc cgtgattggg accgccgtcc
6541 tggagtacat tacacagcag atcgctccaa agaacctgga taatccctct aagaaagagc
6601 aggaactgat cgctaagaaa accgagaagg caaaatatct gagtctggaa acaattaagc
6661 tggcactgga ggagttcaac aagcacaggg atattgacaa acagtgccgc tttgaggaaa
6721 tcctggccaa cttcgcagcc atccccatga tttttgatga gatcgcccag aacaaagaca
6781 atctggctca gatcagtatt aagtaccaga accagggcaa gaaagacctg ctgcaggctt
6841 cagcagaaga tgacgtgaaa gccatcaagg atctgctgga ccagaccaac aatctgctgc
6901 acaagctgaa aatcttccat attagtcagt cagaggataa ggctaatatc ctggataaag
6961 acgaacactt ctacctggtg ttcgaggaat gttacttcga gctggcaaac attgtccccc
7021 tgtataacaa gattaggaac tacatcacac agaagcctta ctctgacgag aagtttaaac
7081 tgaacttcga aaatagtacc ctggccaacg ggtgggataa gaacaaggag cctgacaaca
7141 cagctatcct gttcatcaag gatgacaagt actatctggg agtgatgaat aagaaaaaca
7201 ataagatctt cgatgacaaa gccattaagg agaacaaagg ggaaggatac aagaaaatcg
7261 tgtataagct gctgcccggc gcaaataaga tgctgcctaa ggtgttcttc agcgccaaga
7321 gtatcaaatt ctacaaccca tccgaggaca tcctgcggat tagaaatcac tcaacacata
7381 ctaagaacgg gagcccccag aagggatatg agaaatttga gttcaacatc gaggattgca
7441 ggaagtttat tgacttctac aagcagagca tctccaaaca ccctgaatgg aaggattttg
7501 gcttccggtt ttccgacaca cagagatata actctatcga cgagttctac cgcgaggtgg
7561 aaaatcaggg gtataagctg acttttgaga acatttctga aagttacatc gacagcgtgg
7621 tcaatcaggg aaagctgtac ctgttccaga tctataacaa agatttttca gcatacagca
7681 agggcagacc aaacctgcat acactgtact ggaaggccct gttcgatgag aggaatctgc
7741 aggacgtggt ctataaactg aacggagagg ccgaactgtt ttaccggaag cagtctattc
7801 ctaagaaaat cactcaccca gctaaggagg ccatcgctaa caagaacaag gacaatccta
7861 agaaagagag cgtgttcgaa tacgatctga ttaaggacaa gcggttcacc gaagataagt
7921 tctttttcca ttgtccaatc accattaact tcaagtcaag cggcgctaac aagttcaacg
7981 acgagatcaa tctgctgctg aaggaaaaag caaacgatgt gcacatcctg agcattgacc
8041 gaggagagcg gcatctggcc tactataccc tggtggatgg caaagggaat atcattaagc
8101 aggatacatt caacatcatt ggcaatgacc ggatgaaaac caactaccac gataaactgg
8161 ctgcaatcga gaaggataga gactcagcta ggaaggactg gaagaaaatc aacaacatta
8221 aggagatgaa ggaaggctat ctgagccagg tggtccatga gattgcaaag ctggtcatcg
8281 aatacaatgc cattgtggtg ttcgaggatc tgaacttcgg ctttaagagg gggcgcttta
8341 aggtggaaaa acaggtctat cagaagctgg agaaaatgct gatcgaaaag ctgaattacc
8401 tggtgtttaa agataacgag ttcgacaaga ccggaggcgt cctgagagcc taccagctga
8461 cagctccctt tgaaactttc aagaaaatgg gaaaacagac aggcatcatc tactatgtgc
8521 cagccggatt cacttccaag atctgccccg tgaccggctt tgtcaaccag ctgtacccta
8581 aatatgagtc agtgagcaag tcccaggaat ttttcagcaa gttcgataag atctgttata
8641 atctggacaa ggggtacttc gagttttcct tcgattacaa gaacttcggc gacaaggccg
8701 ctaaggggaa atggaccatt gcctccttcg gatctcgcct gatcaacttt cgaaattccg
8761 ataaaaacca caattgggac actagggagg tgtacccaac caaggagctg gaaaagctgc
8821 tgaaagacta ctctatcgag tatggacatg gcgaatgcat caaggcagcc atctgtggcg
8881 agagtgataa gaaatttttc gccaagctga cctcagtgct gaatacaatc ctgcagatgc
8941 ggaactcaaa gaccgggaca gaactggact atctgattag ccccgtggct gatgtcaacg
9001 gaaacttctt cgacagcaga caggcaccca aaaatatgcc tcaggatgca gacgccaacg
9061 gggcctacca catcgggctg aagggactga tgctgctggg ccggatcaag aacaatcagg
9121 aggggaagaa gctgaacctg gtcattaaga acgaggaata cttcgagttt gtccagaata
9181 gaaataacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat
9241 cctacccata cgatgttcca gattacgctt atccctacga cgtgcctgat tatgcatacc
9301 catatgatgt ccccgactat gcctaaagcc tcgagggtgg tggtagcgat tataaagatg
9361 atgatgataa aggcctgaac gatatctttg aagcccagaa aattgaatgg cacgagtaaa
9421 agcttgtcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
9481 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
9541 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
9601 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
9661 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
9721 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
9781 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
9841 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
9901 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
9961 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
10021 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
10081 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
10141 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
10201 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
10261 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
10321 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
10381 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
10441 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
10501 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
10561 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
10621 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
10681 gagct
//