LOCUS       sequence-74459-e        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6044)
  AUTHORS   Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, 
            Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA
  TITLE     Design of orthogonal genetic switches based on a crosstalk map of 
            sigmas, anti-sigmas, and promoters.
  JOURNAL   Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58.
  PUBMED    24169405
REFERENCE   2  (bases 1 to 6044)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Syst 
            Biol."; date: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pVRa27_1331
FEATURES             Location/Qualifiers
     source          1..6044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             619..636
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(689..707)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      703..750
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      787..1242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(874..893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1084..1105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1269..1373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1374..1442,1443..2234)
                     /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(1592..1611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2405..2993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2894..2913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3147..3164
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3179..3321
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3247..3266)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3407..3429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3423..3614)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4423..5505)
                     /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     5467..5486
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5506..5583)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5789..5808
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5892..5911
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5892..5910
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5911..5935
                     /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             5950..5972
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5991..6008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6018..6041
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgcctccgc ctattggtta ttgtccggca gttggttttg gtggtcgtca tgaacgtagt
       61 gatgcagaac tgctggcagc acatgttgcc ggtgatcgtt atgcatttga tcagctgttt
      121 cgtcgtcatc atcgtcagct gcatcgttta gcacgtctga ccagccgtac cagcgaagat
      181 gcagatgatg cactgcagga tgcaatgctg agcgcacatc gtggtgcagg tagctttcgt
      241 tatgatgcag cagttagcag ctggctgcac cgtattgttg ttaatgcatg tctggatcgt
      301 ctgcgtcgtg caaaagcaca tccgaccgca ccgctggaag atgtttatcc ggttgcagat
      361 cgtaccgcac aggttgaaac cgcaattgca gttcagcgtg cactgatgcg tctgccggtt
      421 gaacagcgtg cagccgttgt tgcagttgat atgcagggtt atagcattgc agataccagt
      481 cgtatgctgg gtgttgcaga aggcaccgtt aaaagccgtt gtgcacgtgc ccgtgcgcgt
      541 ctggctcgcc tgctgggtta tctgaatacc ggtgttaata ttcgtcgcta aggatccaag
      601 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      661 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      721 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      781 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      841 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      961 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1081 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1141 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1201 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg
     1261 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1321 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1381 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1441 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1501 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1561 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1621 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1681 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1741 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1801 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1861 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     1921 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1981 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2041 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2101 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2161 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2221 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2281 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2341 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2401 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2461 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2521 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2581 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2641 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2701 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2761 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2821 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2881 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2941 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3001 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3061 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3121 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3181 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac
     3241 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3301 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3361 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3421 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3481 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3541 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3601 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3661 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3721 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3781 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3841 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3901 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3961 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4021 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4081 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4141 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4201 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4261 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4321 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4381 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4441 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4501 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4561 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4621 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4681 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4741 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4801 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4861 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4921 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4981 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5041 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5101 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5161 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5221 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5281 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5341 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5401 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5461 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5521 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5581 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5641 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5701 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5761 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5821 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     5881 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5941 agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc
     6001 atcatcacag cagcggcctg gaagttctgt tccaggggcc ccat
//
