LOCUS       sequence-74477-e        6017 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 6017)
  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 6017)
  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_375
FEATURES             Location/Qualifiers
     source          1..6017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             592..609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(662..680)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      676..723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      760..1215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(847..866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1057..1078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1242..1346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1347..1415,1416..2207)
                     /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(1565..1584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2378..2966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2867..2886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3120..3137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3152..3294
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3220..3239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3380..3402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3396..3587)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4396..5478)
                     /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     5440..5459
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5479..5556)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5762..5781
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5865..5884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5865..5883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5884..5908
                     /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             5923..5945
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5964..5981
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5991..6014
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgacccagg cagcaagtga tgaaattctg attgcacgta ttgcacaggg tgatcgtctg
       61 gcaatgcagg ttctgtatgg tcgtcatcat gttcgtgttt atcgttttgg tctgcgtctg
      121 gttcgtgatg aacaggcagc agaagatctg attagcgaag tttttctgga tgtttggcgt
      181 caggcaggta aatttgaagg tcgtagcgca gttagcacct ggctgctggc aattacccgt
      241 tttaaagcac tgagcgcact gcgtcgtcgt aaagattttg aactggatga agatgcagcc
      301 aatgcaattg aagataccag cgataatccg gaaaccgttg ttcagaaaaa agatacctct
      361 gaagcactgc gtgaatgtct gaccggtctg agtccggaac atcgtgaaat tgttgatctg
      421 gtgtactatc acgagaaaag cgttgaagaa gtggcagaaa tcgttggtat tccggaaaat
      481 accgttaaaa cccgtctgtt ttatgcccgt aaaaaactgg cagaactgct gaaagcagcc
      541 ggtgttgaac gtggttggcc gtaaggatcc aagcttgcgg ccgcactcga gcaccaccac
      601 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      661 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      721 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      781 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      841 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      901 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      961 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1021 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1081 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1141 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1201 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1261 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1321 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1381 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1441 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1501 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1561 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1621 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1681 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1741 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1801 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1861 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     1921 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1981 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2041 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2101 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2161 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2221 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2281 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2341 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2401 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2461 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2521 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2581 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2641 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2701 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2761 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2821 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2881 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2941 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3001 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3061 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3121 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3181 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     3241 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3301 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3361 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3421 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3481 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3541 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3601 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3661 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3721 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3781 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3841 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3901 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3961 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4021 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4081 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4141 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4201 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4261 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4321 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4381 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4501 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4561 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4621 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4681 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4741 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4801 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4861 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4921 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4981 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5041 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5101 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5161 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5221 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5281 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5341 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5401 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5461 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5521 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5581 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5641 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5701 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5761 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5821 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     5881 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     5941 ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc ctggaagttc
     6001 tgttccaggg gccccat
//
