LOCUS       sequence-74487-e        6014 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 6014)
  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 6014)
  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: pVRa37_2513
FEATURES             Location/Qualifiers
     source          1..6014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             589..606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(659..677)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      673..720
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      757..1212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(844..863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1054..1075
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1239..1343
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1344..1412,1413..2204)
                     /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(1562..1581)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2375..2963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2864..2883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3117..3134
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3149..3291
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3217..3236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3377..3399
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3393..3584)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4393..5475)
                     /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     5437..5456
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5476..5553)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5759..5778
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5862..5881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5862..5880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5881..5905
                     /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             5920..5942
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5961..5978
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5988..6011
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggcaagcg ataaagaact ggcagatttt ctggcaggcg ttgaacgtcg tgcatttaaa
       61 caggcagcct atgcagttcg tgatgatgat gcaagcctgg atattgttca ggatgcaatg
      121 attcgtctgg ccgaaaaata tggtgatcgt ccggcagcag aactgccgct gctgtttcag
      181 cgtatcctgc agaatgcaat ccatgattat tttcgtcgtc agaaagtgcg taatacctgg
      241 gttagcctgt ttagcagcct gaataatacc gatgatgacg aatttgatcc gctggaaacc
      301 ctggaaagcg cagatggtga tggtgttgaa agcggtgaaa cccgtctgga acgtgaacag
      361 gttctggcac tgattgatga tgaaattcag aaactgcctg cacgtcagcg tgaagcattt
      421 ctgatgcgtt attgggaaga tatggatgtt gcagaaaccg cagcagcaat gggttgtagc
      481 gaaggtagcg ttaaaaccca ttgtagccgt gcaacccatg cactggcaag cgcactgaaa
      541 gcaaaaggta ttaccctgta aggatccaag cttgcggccg cactcgagca ccaccaccac
      601 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      661 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      721 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      781 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      841 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      901 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      961 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1021 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1081 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1141 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1201 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1261 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1321 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1381 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1441 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1501 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1561 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     1621 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1681 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1741 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1801 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1861 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     1921 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1981 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2041 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2101 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2161 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2221 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2281 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2521 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3061 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3121 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3181 gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata
     3241 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3301 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3361 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3421 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3481 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3541 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3601 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3661 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3721 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3781 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3841 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3901 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3961 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4021 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4081 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4141 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4201 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4261 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4321 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4381 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4441 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4501 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4561 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4621 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4681 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4741 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4801 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4861 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4921 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4981 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5041 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5101 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5161 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5221 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5281 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5341 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5401 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5461 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5521 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5581 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5641 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5701 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5761 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5821 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     5881 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     5941 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gaagttctgt
     6001 tccaggggcc ccat
//
