LOCUS       sequence-74457-e        5993 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 5993)
  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 5993)
  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: pVRa26_837
FEATURES             Location/Qualifiers
     source          1..5993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             568..585
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(638..656)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      652..699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      736..1191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(823..842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1033..1054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1218..1322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1323..1391,1392..2183)
                     /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(1541..1560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2354..2942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2843..2862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3096..3113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3128..3270
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3196..3215)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3356..3378
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3372..3563)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4372..5454)
                     /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     5416..5435
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5455..5532)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5738..5757
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5841..5860
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5841..5859
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5860..5884
                     /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             5899..5921
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5940..5957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5967..5990
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggaagcca atatttttag cgatcaggca tttcgtgcag aactgggtca gcatctggca
       61 cgtctgtggc gttatggtct gctgctgagc cgtaatcgtc atgttgcaga agatctggtt
      121 caggcaacct gtgttcgtgc actggaacgt agcggtcagt atgttgcagg cacccgtatg
      181 gatcgttggc tgctgagtat tctgcatagc atttggctga atgaagttcg tgcccgtcgt
      241 gttcgtcagg gtcagggtgt tgcagatgca gatagccagc tgacctttga tggtgaatat
      301 gcagcacaga cccatgttat ggcagcacag gttattcgtc gtgttgatgc actgccggaa
      361 acccagcgtg aaaccgttta tctggcctat gttgaaggtc tgagttatcg tgaagttgcc
      421 gaaattctgc aggttccgat tggcaccgtt atgagccgtc tggcaaccgc acgtctgaaa
      481 ctggcagaat atccgcctct gcaggcagtt ccgaatagtc cggcaggcga tcgtcagtaa
      541 ggatccaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
      601 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      661 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      721 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      781 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      841 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      901 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      961 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1021 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1081 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1141 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca
     1201 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1261 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1321 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1381 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1441 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1501 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1561 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1621 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1681 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1741 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1801 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1861 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1921 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1981 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2041 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2101 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2161 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     2221 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2281 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2341 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2401 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2461 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2521 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2581 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2641 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2701 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2761 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2821 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2881 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2941 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3001 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3061 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3121 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     3181 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3241 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3301 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3361 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3421 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3481 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3541 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3601 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3661 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3721 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3781 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3841 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3901 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3961 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4021 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4081 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4141 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4201 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4261 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4321 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4381 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4441 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4501 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4561 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4621 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     4681 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4741 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4801 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4861 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4921 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4981 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5041 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5101 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5161 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5221 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5281 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5341 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5401 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5461 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5521 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5581 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5641 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5701 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5761 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     5821 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5881 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     5941 atcatcatca tcatcacagc agcggcctgg aagttctgtt ccaggggccc cat
//
