LOCUS Exported 9289 bp ds-DNA circular SYN 09-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..9289
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 3..27
/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 42..64
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 74..94
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 95..4198
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4199..4216
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
terminator 4331..4378
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 4415..4870
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(4985..5845)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5846..5938)
/gene="bla"
/label=AmpR promoter
rep_origin 6019..6607
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 6793..6935
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(7037..7228)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(7800..8882)
/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(8883..8960)
/gene="lacI"
/label=lacI promoter
promoter 9273..2
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
61 gagatatacc atgcccaaga agaagaggaa ggtgatggat aagaaatact caataggctt
121 agatatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc
181 taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg
241 ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg
301 tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga
361 gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga
421 agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca
481 tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc
541 ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt
601 gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt
661 acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa
721 agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct
781 ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac
841 ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga
901 tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt
961 gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa
1021 tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca
1081 tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga
1141 aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca
1201 agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt
1261 attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc
1321 tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt
1381 ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc
1441 ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc
1501 tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca
1561 atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc
1621 aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata
1681 tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt
1741 tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt
1801 caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc
1861 ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa
1921 tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag
1981 ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa
2041 acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg
2101 tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc
2161 caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca
2221 aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg
2281 tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa
2341 agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac
2401 aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa
2461 agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga
2521 aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga
2581 tattaatcgt ttaagtgatt atgatgtcga tcacattgtt ccacaaagtt tccttaaaga
2641 cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa
2701 cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc
2761 caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag
2821 tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa
2881 gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact
2941 tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga
3001 tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct
3061 aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt
3121 ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat
3181 aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga
3241 aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac
3301 tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat
3361 gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc
3421 aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa
3481 aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt
3541 ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat
3601 ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga
3661 agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg
3721 tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc
3781 aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc
3841 agaagataac gaacaaaaac aattgtttgt tgagcagcat aagcattatt tagatgagat
3901 tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa
3961 agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat
4021 tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac
4081 aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca
4141 tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacca
4201 tcaccaccac catcactaat gactcgagtc tggtaaagaa accgctgctg cgaaatttga
4261 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
4321 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
4381 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
4441 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
4501 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
4561 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
4621 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
4681 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
4741 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
4801 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
4861 tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc
4921 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
4981 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
5041 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
5101 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
5161 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
5221 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
5281 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
5341 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
5401 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
5461 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
5521 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
5581 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
5641 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
5701 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
5761 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
5821 cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat
5881 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
5941 ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
6001 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
6061 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
6121 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
6181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
6241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
6301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
6361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
6421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
6481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
6541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
6601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
6661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
6721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
6781 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
6841 catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
6901 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
6961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
7021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
7081 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
7141 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
7201 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
7261 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
7321 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
7381 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
7441 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
7501 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
7561 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
7621 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
7681 tcatgctagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
7741 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
7801 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
7861 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
7921 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
7981 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
8041 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
8101 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
8161 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
8221 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
8281 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
8341 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
8401 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
8461 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
8521 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
8581 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
8641 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
8701 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
8761 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
8821 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
8881 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
8941 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
9001 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
9061 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
9121 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
9181 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
9241 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactata
//