LOCUS Exported 6076 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET21a-Ef0113 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6076) AUTHORS Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA TITLE Genomic mining of prokaryotic repressors for orthogonal logic gates. JOURNAL Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411. PUBMED 24316737 REFERENCE 2 (bases 1 to 6076) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6076 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" promoter 494..598 /gene="bla" /label=AmpR promoter CDS 599..1459 /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" primer_bind complement(817..836) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1630..2218 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2119..2138 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2372..2389 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2404..2546 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2472..2491) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2632..2654 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2648..2839) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3648..4730) /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" primer_bind 4692..4711 /label=LacI-R /note="LacI, reverse primer" promoter complement(4731..4808) /gene="lacI" /label=lacI promoter primer_bind 5014..5033 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5117..5136 /label=T7 /note="T7 promoter, forward primer" promoter 5117..5135 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5136..5160 /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 5174..5196 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5215..5232 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5242..5265 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 5920..5937 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5990..6008) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6004..6051 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3301 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3361 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3421 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3481 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3541 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3601 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3721 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3781 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3841 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3901 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3961 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4021 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4081 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4141 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4201 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4261 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4321 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4381 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4441 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4501 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4561 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4621 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4681 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4741 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4801 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4861 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4921 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4981 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5041 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5101 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5161 cctctagaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat 5221 catcatcatc acagcagcgg cctggaagtt ctgttccagg ggccccatat gagcatcaac 5281 aaaaaagaca tcaaaaaaga agagatcatc tttaccgcca tgaaacactt tagcaaattc 5341 ggctacgaaa aaaccaaact gctggatatt gccaatgaag caaaaaccag caccaccacc 5401 gtttatagct ttttcaaaac caaacacgat ctgtatgaag ccgtgattgt gtataacctg 5461 gatattatca acagcgaact ggaaaaaatc gccgataaaa gcagcaccat cgatgaatat 5521 atcatgaaca ttattgaacg cgtgcgcctg aaaagcgata aagaacagct gctgctgaac 5581 ttttatatgc tgattagcac caatagcgtg cagattaaaa acctggaaat catcgaaaaa 5641 tttgaacgca aacgcttcag ctattacctg aaatttatcg agaacccgaa aatcaaagat 5701 cgttataccc tgctgttcat cgataacatt atcaccatct acatctacag ctacttcaac 5761 gtgttttatc gcaaaaaaat ctttctgtat ctgagcctgt atcagcagga tcgtgaaagc 5821 attgaatttg aagaggatct gatgaaatat ctgaaactgc tggtgaaaaa acgcctgctg 5881 gaaattgaat aaggatccaa gcttgcggcc gcactcgagc accaccacca ccaccactga 5941 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 6001 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga 6061 ggaactatat ccggat //