LOCUS       sequence-74059-e        5453 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5453)
  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 5453)
  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: pVRa32_3724
FEATURES             Location/Qualifiers
     source          1..5453
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             28..45
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(98..116)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      112..159
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      196..651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(283..302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     493..514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        678..782
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(783..851,852..1643)
                     /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(1001..1020)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1814..2402
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2303..2322
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2556..2573
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2588..2730
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2656..2675)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2816..2838
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2832..3023)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3832..4914)
                     /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     4876..4895
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4915..4992)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5198..5217
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5301..5320
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5301..5319
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5320..5344
                     /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             5359..5381
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5400..5417
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5427..5450
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 ggatccaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
       61 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      121 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      181 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      241 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      541 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      601 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca
      661 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      721 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      781 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      841 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      901 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
      961 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1021 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1081 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1141 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1201 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1261 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1321 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1381 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1441 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1501 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1561 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1621 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1681 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1741 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1801 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1861 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1921 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     1981 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2041 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2101 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2161 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2221 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2281 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2341 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2401 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2461 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2521 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2581 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     2641 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2701 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2761 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2821 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2881 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2941 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3001 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3061 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3121 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3181 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3241 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3301 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3361 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3421 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3481 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3541 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3601 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3661 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     3721 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3781 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     3841 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3901 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3961 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4021 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4081 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     4141 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4201 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4261 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4321 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4381 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4441 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4501 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4561 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4621 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4681 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     4741 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     4801 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     4861 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     4921 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     4981 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5041 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5101 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5161 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5221 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     5281 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5341 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     5401 atcatcatca tcatcacagc agcggcctgg aagttctgtt ccaggggccc cat
//
