LOCUS       sequence-74430-e        5969 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 5969)
  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 5969)
  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: pVRa07_1134
FEATURES             Location/Qualifiers
     source          1..5969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             544..561
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(614..632)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      628..675
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      712..1167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(799..818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1009..1030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1194..1298
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1299..1367,1368..2159)
                     /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(1517..1536)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2330..2918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2819..2838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3072..3089
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3104..3246
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3172..3191)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3332..3354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3348..3539)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4348..5430)
                     /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     5392..5411
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5431..5508)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5714..5733
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5817..5836
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5817..5835
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5836..5860
                     /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             5875..5897
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5916..5933
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5943..5966
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggatgcac cgctggcaga acaggatctg aaagcactgt ttctgcgtca tgcacgtcag
       61 ctgcagagct atctgctgcg taaaacccgt gatccgcatc tggcagcaga tctggcacaa
      121 gaaagttttc tgcgcctggc cgaacagcgt cataaagaac cgattgaaca tgttgatgcc
      181 tatctgtatc gcaccgcaca taatctgacc attgatcatt atcgtcaaga acgtcgtcgt
      241 cgtaccgaaa cccagccgaa tgatgatctg gcaggttttg ttgcagatca gcctggtccg
      301 gaagaaagcg cagttgatgc agatgcactg gcacgtctgc gtgaaattgt tgccgaactg
      361 cctccgcgta cccgtgaaat ttttcgtctg aatcgtctgg aaggtctgac ccatgcagaa
      421 gttgcacgtc gcctggaaat tagcgatagc agcgttcaga aacatctggc acgtaccctg
      481 gcacatgtta tgcaggcact ggaagatagc cgttaaggat ccaagcttgc ggccgcactc
      541 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      601 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      661 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      721 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      781 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      841 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      901 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      961 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1021 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1081 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1141 ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     1201 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1261 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1321 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1381 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1441 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1501 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1561 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1621 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1681 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1741 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1801 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     1861 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1921 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1981 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2041 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2101 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2161 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2221 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2281 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2341 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2401 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2461 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2521 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2581 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2641 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2701 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2761 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2821 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2881 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2941 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3001 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3061 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3121 tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg
     3181 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3241 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3301 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3361 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3421 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3481 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3541 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3601 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3661 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3721 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3781 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3841 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3901 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3961 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4021 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4081 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4141 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4201 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4261 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4321 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4381 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4441 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4501 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4561 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4621 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4681 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4741 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4801 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4861 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4921 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4981 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5041 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5101 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5161 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5221 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5281 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5341 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5401 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5461 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5521 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5581 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5641 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5701 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5761 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     5821 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     5881 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
     5941 gcctggaagt tctgttccag gggccccat
//
