LOCUS       sequence-74453-e        5951 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 5951)
  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 5951)
  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: pVRa22_1147
FEATURES             Location/Qualifiers
     source          1..5951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             526..543
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(596..614)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      610..657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      694..1149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(781..800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     991..1012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1176..1280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1281..1349,1350..2141)
                     /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(1499..1518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2312..2900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2801..2820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3054..3071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3086..3228
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3154..3173)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3314..3336
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3330..3521)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4330..5412)
                     /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     5374..5393
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5413..5490)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5696..5715
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5799..5818
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5799..5817
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5818..5842
                     /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             5857..5879
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5898..5915
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5925..5948
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgccgctgg ataccgatct gcatacccgt tttagcgcac tgctgcaaca gcatcgtgca
       61 attgttctga aagttgcagc aagctattgt cgtgatcgtg atgaccgtga tgaactggca
      121 caagaaattg caacccagct gtggcgtgca tttccgggtt atgatccgag ccgtcgtttt
      181 agcacctgga tgtatcgtat tgcactgaat gttgcaatta gcgatctgcg tggtcgtcgt
      241 gcagccggtg aaccgattgc actggatgca gttgttgaac gtattgcaga tccgtgtgca
      301 catgatccgg ttcgtgaaca gcaggttgca gcactgtatg catttattgc acatcagctg
      361 cctctggaac gtgccctgct gctgctgtat ctggatcagc atagctatcg tgaaattgca
      421 gatgttctgg gtattagcga aaccaatgtt gccaccaaac tgagccgtct gaaaagccgt
      481 attcgtgcag aactgtaagg atccaagctt gcggccgcac tcgagcacca ccaccaccac
      541 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      601 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      661 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      721 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      781 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      841 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      901 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      961 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1021 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1081 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     1141 ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1201 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1261 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1321 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1381 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1441 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1501 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1561 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1621 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1681 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1741 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1801 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     1861 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     1921 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     1981 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2041 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2101 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2161 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2221 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2281 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2341 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2401 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2461 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2521 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2581 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2641 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2701 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2761 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2821 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2881 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2941 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3001 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3061 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3121 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3181 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3241 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3301 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3361 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3421 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3481 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3541 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3601 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3661 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     3721 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     3781 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     3841 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     3901 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     3961 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4021 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4081 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4141 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4201 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4261 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4441 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4501 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4561 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     4621 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4681 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     4741 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     4801 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     4861 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     4921 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     4981 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5041 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5101 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5161 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5221 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5281 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5341 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5401 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5461 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     5521 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     5581 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     5641 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     5701 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     5761 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     5821 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     5881 ataccatggg cagcagccat catcatcatc atcacagcag cggcctggaa gttctgttcc
     5941 aggggcccca t
//
