LOCUS       sequence-74492-e        5960 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 5960)
  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 5960)
  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: pVRa39_2973
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             535..552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(605..623)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      619..666
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      703..1158
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(790..809)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1000..1021
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1185..1289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1290..1358,1359..2150)
                     /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(1508..1527)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2321..2909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2810..2829
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3063..3080
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3095..3237
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3163..3182)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3323..3345
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3339..3530)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4339..5421)
                     /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     5383..5402
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5422..5499)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5705..5724
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5808..5827
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5808..5826
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5827..5851
                     /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             5866..5888
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5907..5924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5934..5957
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgggtcgtg aaagcaaagc agaacgtgat gcagcattta ccgcatttat gaccgatgca
       61 accccgagcc tgctgcgtac cgcatggctg ctgaccggta gcaccgatcg tagccaagaa
      121 ctggttcagg cagcatttgt taaaacctat gcagcatggc cgaaagttcg tcgtgaatca
      181 gcactggcct atgcacgtcg tgttctggtt aatcagaaaa ccgatacctg gcgtaaaacc
      241 agccgtgaag ttattgttgg tgaagtgccg gaacaagaaa ttcgtccgga taataaagtg
      301 gaacatcgtg atcagattct gcgtatgctg gatctgctgc ctcgtgcaca gcgtaccatt
      361 gttgttctgc gttattatca ggatctgagc gaacagcagg ttgcagatgt tctgggtatt
      421 agcgttggta gcgttaaaag cgcaggtagc cgtggtctgg caaccctgcg caccgcacat
      481 ctgagcaccg aaggtagcca gcgttaagga tccaagcttg cggccgcact cgagcaccac
      541 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      601 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      661 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      721 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      781 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      841 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      901 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      961 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1021 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1081 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1141 tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1201 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1261 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1321 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1381 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1441 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1501 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1561 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1621 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1681 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1741 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1801 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     1861 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1921 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1981 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2041 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2101 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2161 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2221 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2281 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2341 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2401 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2461 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2521 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2581 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2641 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2701 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2761 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2821 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2881 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2941 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3001 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3061 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3121 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     3181 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3241 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3301 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3361 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3421 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3481 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3541 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3601 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3661 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3721 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3781 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3841 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3901 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3961 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4021 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4081 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4141 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4201 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4261 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4321 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4381 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4441 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4501 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4561 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4621 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4681 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4741 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4801 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4861 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4921 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4981 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5041 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5101 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5161 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5221 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5281 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5341 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5401 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5461 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5521 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5581 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5641 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5701 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5761 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     5821 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5881 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggaag
     5941 ttctgttcca ggggccccat
//
