LOCUS       sequence-74464-e        6164 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 6164)
  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 6164)
  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_4265
FEATURES             Location/Qualifiers
     source          1..6164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             739..756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(809..827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      823..870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      907..1362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(994..1013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1204..1225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1389..1493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1494..1562,1563..2354)
                     /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(1712..1731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2525..3113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3014..3033
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3267..3284
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3299..3441
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3367..3386)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3527..3549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3543..3734)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4543..5625)
                     /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     5587..5606
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5626..5703)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5909..5928
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6012..6031
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6012..6030
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6031..6055
                     /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             6070..6092
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6111..6128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6138..6161
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggcaggcg gtgcaagcca tgatggtgtt agcgatcaag aactgctggc acgtcatgtt
       61 gaaggtgatc cggatgcatt tggtgaactg gttcgtcgtc atcgtgatcg tctgtgggca
      121 gttgcactgc gtaccctggg tgatcgtgaa gaggcagcag atgcagttca ggatgcactg
      181 gttagcgcat atcgtgcagc acataccttt cgtggtcagg cagcagttac cacctggctg
      241 catcgtatta ccgttaatgc atgtctggat cgtgcacgta aagcagcaag ccgtaaaacc
      301 gcaccggttg atgataccga acgtctggaa cagctgctgg aaccgcatga aagcgcaagc
      361 gcaccggcag aacgtaatga tctgcatcgt caactgctgg aagcactggg caccctgcct
      421 gcagatcagc gtgcagccct ggttctggtt gatatgcagg gttatccggt tgcagaagca
      481 gcacgtattc tggatgttcc ggcaggcacc attaaaagcc gttgtgcacg tggtcgtgcc
      541 cgtctgctgc cgctgctgac acatctgcgt ccggatggta gcggtgaaag tcgtaaaccg
      601 ggtccggaac gtaatcgtac ccatggtgca agcgttccgc ctgcagcagg tccgagtgaa
      661 gccggtccgg gtgatagcac cgcagttaaa ggtggtggtg gtcgcgcata aggatccaag
      721 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      781 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      841 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      901 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      961 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1021 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1081 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1141 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1201 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1261 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1321 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg
     1381 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1441 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1501 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1561 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1621 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1681 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1741 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1801 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1861 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1921 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1981 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2041 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2101 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2161 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2221 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2281 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2341 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2401 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2461 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2521 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2581 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2641 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2701 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2761 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2821 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2881 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2941 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3001 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3061 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3121 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3181 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3241 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3301 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac
     3361 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3421 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3481 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3541 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3601 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3661 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3721 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3781 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3841 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3901 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3961 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4021 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4081 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4141 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4201 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4261 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4321 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4381 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4441 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4501 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4561 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4621 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4681 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4741 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4801 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4861 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4921 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4981 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5041 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5101 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5161 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5221 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5281 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5341 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5401 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5461 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5521 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5581 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5641 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5701 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5761 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5821 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5881 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5941 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     6001 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     6061 agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc
     6121 atcatcacag cagcggcctg gaagttctgt tccaggggcc ccat
//
