LOCUS       sequence-74466-e        6080 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 6080)
  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 6080)
  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: pVRa29_371
FEATURES             Location/Qualifiers
     source          1..6080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             655..672
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(725..743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      739..786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      823..1278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(910..929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1120..1141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1305..1409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1410..1478,1479..2270)
                     /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(1628..1647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2441..3029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2930..2949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3183..3200
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3215..3357
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3283..3302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3443..3465
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3459..3650)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4459..5541)
                     /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     5503..5522
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5542..5619)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5825..5844
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5928..5947
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5928..5946
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5947..5971
                     /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             5986..6008
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6027..6044
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6054..6077
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgcgtctgg ttcgtcgtgc actggcacgt gatgccggtg catttcgtac cattattaaa
       61 acccataacc agcgtctgta tcgtctggca cgcggtattc tgcgtaatga tgccgaagca
      121 gaagatgttg ttcaagaggc ctatgttcgt gcatttgcca atctggcagc atttcgtggt
      181 gatgcaagcc tggcaacctg gctgagccgt attgttatta atgaagcact gggtcgtctg
      241 cgtaaacgtc gtcgtaccgt tgcaatgccg gaaagtccgg aagcacaggt tattcgtttt
      301 ccgctgaatc cgagtgatga tccggaacgt accatggcac agcgtcagat tctggcactg
      361 gttgaacgtg caaccgatag cctgccggat atttatcgta tggtttttgt tgcccgtgtg
      421 attgaaggtc tgagcatgga agaaaccgct gatctgctgg gtgttcgtcc ggaaaccgtt
      481 aaaacccgtc tgcatcgtgc acgtgccctg ctgcgtaaag cactggatga tgaaattggt
      541 ccggttctgc tggatgcatt tccgtttgca ggtcgtcgtt gtggtcgtct gacccgtgca
      601 gttatgggtc gcctgggtct ggaataagga tccaagcttg cggccgcact cgagcaccac
      661 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      721 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      781 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      841 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      901 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      961 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1021 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1081 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1141 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1201 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1261 tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1321 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1381 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1441 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1501 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1561 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1621 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1681 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1741 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1801 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1861 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1921 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     1981 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2041 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2101 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2161 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2221 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2281 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2341 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2401 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2461 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2521 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2581 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2641 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2701 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2761 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2821 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2881 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2941 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3001 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3061 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3121 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3181 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3241 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     3301 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3361 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3421 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3481 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3541 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3601 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3661 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3721 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3781 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3841 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3901 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3961 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4021 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4081 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4141 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4201 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4261 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4321 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4381 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4441 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4501 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4561 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4621 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4681 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4741 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4801 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4861 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4921 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4981 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5041 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5101 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5161 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5221 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5281 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5341 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5401 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5461 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5521 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5581 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5641 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5701 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5761 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5821 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5881 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     5941 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     6001 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggaag
     6061 ttctgttcca ggggccccat
//
