LOCUS       sequence-74469-e        5990 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 5990)
  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 5990)
  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: pVRa30_83
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             565..582
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(635..653)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      649..696
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      733..1188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(820..839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1030..1051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1215..1319
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1320..1388,1389..2180)
                     /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(1538..1557)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2351..2939
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2840..2859
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3093..3110
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3125..3267
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3193..3212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3353..3375
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3369..3560)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4369..5451)
                     /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     5413..5432
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5452..5529)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5735..5754
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5838..5857
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5838..5856
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5857..5881
                     /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             5896..5918
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5937..5954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5964..5987
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgctggaac tgattagctt ttttaacaaa aacagcttta acaaagtgag ctataaaaaa
       61 ctgaccaaag aacgtctgag ccagaatttt gaactgctga ttaaagaaaa tctgaccagc
      121 atgtatcgtg tggcaaaagg tattctgaaa gtggaagagg atatcgaaga tgcaattcag
      181 accaccattc tgatcgcctt caacaaaatt aacaccctga aagatgaacg ctatttcaaa
      241 acctggctga ttcgcattct gattaacgaa tgcaacaaaa tctacaaaaa aaacaaaaat
      301 accccgaaag aactgagcag cgaactgtat attaccgaaa actatgagaa tattgatctg
      361 tatgatgcca ttaaatgcct gaaagaagat ctgcgcgtta ttaccatcct gttttatttt
      421 gaggatatga gctacaaaga tattgccgtt gcactgaata ttgcagaagg caccgttaaa
      481 agccgtctgt ttcgtgcaaa agaagcactg tacgagatcc tgaaagacaa catttaagga
      541 tccaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
      601 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
      661 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
      721 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1141 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
     1201 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1261 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1321 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1381 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1441 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1501 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1561 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1621 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1681 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1741 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1801 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1861 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1921 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1981 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2041 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2101 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2161 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2221 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2281 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2341 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2401 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2461 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2521 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2581 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2641 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2701 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2761 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2821 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2881 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2941 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3001 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3061 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3121 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg
     3181 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3241 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3301 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3361 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3421 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3481 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3541 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3601 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3661 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     3721 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     3781 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3841 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3901 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     3961 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4021 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4081 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4141 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4201 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4261 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4321 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4381 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4441 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4501 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4561 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4621 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     4681 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     4741 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     4801 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4861 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4921 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     4981 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5041 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5101 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5161 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5221 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5281 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5341 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5401 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5461 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5521 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5581 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     5641 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     5701 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     5761 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     5821 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     5881 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     5941 atcatcatca tcacagcagc ggcctggaag ttctgttcca ggggccccat
//
