LOCUS       sequence-74435-e        6098 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 6098)
  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 6098)
  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: pVRa12_807
FEATURES             Location/Qualifiers
     source          1..6098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             673..690
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(743..761)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      757..804
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      841..1296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(928..947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1138..1159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1323..1427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1428..1496,1497..2288)
                     /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(1646..1665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2459..3047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2948..2967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3201..3218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3233..3375
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3301..3320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3461..3483
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3477..3668)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4477..5559)
                     /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     5521..5540
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5560..5637)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5843..5862
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5946..5965
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5946..5964
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5965..5989
                     /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             6004..6026
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6045..6062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6072..6095
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgtttgatc tgcgtcagaa tgatctgacc cgtgatgaat ttcaagaact ggcactgaaa
       61 catatcgatc cgctgtatag cgcagcactg cgtctgacca aaaatgatcg tgatgcagaa
      121 gatctggtcc aggatacctt tctgcgtgca tatcgctttt tcgataaatt tgaacgtggc
      181 accaacatta aagcctggct gtttaaaatt ctgaccaaca ccttcattaa ccgttatcgt
      241 cgtagcgtga aagaacgtaa tattgttgaa ggtagcgaac gtgatgccgt gcatgaacgt
      301 tttgttagcc gtgaagcagc agaatatgca gcaaatccgg aacagtattt ttttgatcgt
      361 ctgctgagtg atgatgttct gaaagcagtt gatagcctgc cgattgattt tcgtatggtt
      421 gttattctgg ccgatctgca agagtttagc tataaagaaa tcgccgaaat tctggatgtt
      481 ccggttggca ccgttatgag ccgtctgtat cgtggtcgtc gcctgctgca gaaagcactg
      541 gcaagctatg cagttgttag cggtgttatt cagctgccgg aatcagatga tggtgaaagt
      601 attgatctgg aaacctatcg tcgtcgccgt ggtaaaagcg cataaggatc caagcttgcg
      661 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      721 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      781 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
      841 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      901 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      961 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1021 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1081 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1141 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1201 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1261 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     1321 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1381 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1441 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1501 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1561 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1621 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1681 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1741 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1801 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1861 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1921 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     1981 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2041 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2101 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2161 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2221 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2281 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2581 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag
     3301 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3361 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3421 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3481 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3541 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3601 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3661 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3721 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3781 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     3841 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     3901 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3961 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4021 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4081 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4141 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4201 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4261 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4321 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4381 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4441 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4501 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4561 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4621 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4681 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4741 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4801 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4861 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4921 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4981 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5041 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5101 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5161 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5221 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5281 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5341 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5401 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5461 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5521 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5581 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5641 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5701 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5761 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5821 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5881 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5941 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     6001 aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc
     6061 acagcagcgg cctggaagtt ctgttccagg ggccccat
//
