LOCUS       sequence-74482-e        6023 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 6023)
  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 6023)
  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: pVRa33_423
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             598..615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(668..686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      682..729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      766..1221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(853..872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1063..1084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1248..1352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1353..1421,1422..2213)
                     /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(1571..1590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2384..2972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2873..2892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3126..3143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3158..3300
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3226..3245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3386..3408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3402..3593)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4402..5484)
                     /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     5446..5465
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5485..5562)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5768..5787
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5871..5890
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5871..5889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5890..5914
                     /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             5929..5951
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5970..5987
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5997..6020
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgagcacca aacaggcagc aaccgatgat gttctgattg cacgtattgc acagggtgat
       61 cgtgttgcaa tgcaggttct gtatggtcgt catcatgtta aagtgtttcg ttttggtctg
      121 cgtctggttc gtaatgagca gattgcagaa gatctgatta gcgaagtttt tctggatgtt
      181 tggcgtcagg caggtaaatt tgaaggtcgt agcagcgtta gcacctggct gctggcaatt
      241 acccgtttta aagcactgag cgcactgcgt cgtcgtaaag atgcagaact ggatgatgaa
      301 gcagcagcag ccattgaaga tccgagtgat gatccggaaa ttgcagttca gaaaaaagat
      361 accggtgaag cactgcgtaa atgtctgagc agcctgacac cggaacatcg tgaaattgtt
      421 gatctggtgt actatcacga gaaaagcatt gaagaagtgg cagaaatcgt tggtattccg
      481 gaaaataccg ttaaaacccg tctgttttat gcccgtaaaa aactggccga tctgctgcag
      541 gcagccggtg ttcagcgtgg ttggccgtaa ggatccaagc ttgcggccgc actcgagcac
      601 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      661 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      721 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      841 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      961 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1081 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1141 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1201 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1261 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1321 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1381 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1441 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1501 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1561 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1621 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1681 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1741 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1801 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1861 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1921 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1981 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2041 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2101 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2161 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2221 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2281 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2341 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2401 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2461 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2521 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2581 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2641 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2701 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2761 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2821 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2881 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2941 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3001 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3061 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3121 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3181 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     3241 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3301 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3361 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3421 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3481 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3541 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3601 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3661 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3721 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3781 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3841 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3901 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3961 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4021 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4081 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4141 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4201 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4261 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4321 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4381 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4441 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4501 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4561 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4621 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4681 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4741 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4801 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4861 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4921 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4981 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5041 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5101 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5161 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5221 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5281 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5341 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5401 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5461 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5521 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5581 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5641 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5701 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5761 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5821 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     5881 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     5941 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg
     6001 aagttctgtt ccaggggccc cat
//
