LOCUS       sequence-74422-e        6032 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 6032)
  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 6032)
  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: pVRa02_915
FEATURES             Location/Qualifiers
     source          1..6032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             607..624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(677..695)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      691..738
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      775..1230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(862..881)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1072..1093
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1257..1361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1362..1430,1431..2222)
                     /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(1580..1599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2393..2981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2882..2901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3135..3152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3167..3309
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3235..3254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3395..3417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3411..3602)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4411..5493)
                     /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     5455..5474
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5494..5571)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5777..5796
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5880..5899
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5880..5898
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5899..5923
                     /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             5938..5960
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5979..5996
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6006..6029
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgagcggtc agaaaagcga tcaagaactg gttgaactgg tgcagcaggg tgataaaaat
       61 gcctttaatc tgctggttct gcgttatcag agccgtgtta ttaatctgat tagccgttat
      121 gttcgcaatc aggcagatgt tgccgatgtt gcacaagaag catttattaa agcatatcgt
      181 gccctggcaa attttcgtgg tgaaagcgca ttttatacct ggctgtatcg tattgcagtg
      241 aataccgcca aaaattatct ggttagccag ggtcgtcgtg caccggcaaa tgatgttgat
      301 gccgatgatg cagaatatta tgatggtagt gatgccctga aagaatttgc aagtccggaa
      361 cgtctgatgc tgagtgatga aattaaacgt gtggtgtttg aaaccctgga agaactgcct
      421 gaagaactga aaatggcaat tagcctgcgt gaactggatg gtatgagcta tgaagatatt
      481 gccaacgtta tggaatgtcc ggttggcacc gttcgtagcc gtatttttcg tgcacgtgaa
      541 gttattgatc gtaaactgca gccgctgctg gaaagctaag gatccaagct tgcggccgca
      601 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      661 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      721 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      781 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      841 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      901 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      961 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1021 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1081 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1141 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1201 attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     1261 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1321 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1381 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1441 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1501 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1561 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1621 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1681 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1741 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1801 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1861 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     1921 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1981 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2041 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2101 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2161 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2221 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2281 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2341 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2521 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3061 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3121 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3181 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     3241 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3301 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3361 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3421 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3481 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3541 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3601 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3661 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3721 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3781 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3841 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3901 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3961 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4021 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4081 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4141 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4201 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4261 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4321 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4381 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4501 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4561 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4621 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4681 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4741 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4801 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4861 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4921 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4981 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5041 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5101 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5161 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5221 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5281 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5341 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5401 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5461 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5521 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5581 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5641 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5701 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5761 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5821 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     5881 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     5941 gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca
     6001 gcggcctgga agttctgttc caggggcccc at
//
