LOCUS       sequence-74425-e        6020 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 6020)
  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 6020)
  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: pVRa03_1244
FEATURES             Location/Qualifiers
     source          1..6020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             595..612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(665..683)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      679..726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      763..1218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(850..869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1060..1081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1245..1349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1350..1418,1419..2210)
                     /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(1568..1587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2381..2969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2870..2889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3123..3140
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3155..3297
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3223..3242)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3383..3405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3399..3590)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4399..5481)
                     /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     5443..5462
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5482..5559)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5765..5784
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5868..5887
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5868..5886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5887..5911
                     /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             5926..5948
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5967..5984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5994..6017
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggcagata aactgctgat tagcgaagaa gaactgctgg aagaactgcg tgatccggca
       61 cgtcgtcgta tggcatttga aaaagttgtg cgtctgtata accgcaaact gtattggcag
      121 attcgtcgca tggttctgag ccatgatgat accgatgatc tgctgcagaa cacctttatg
      181 aaagcatggt cagcactgga tggttttcgt ggtgaagcaa aactgagcac ctggctgtat
      241 cgtattgcaa tgtatgaaag cctgaatttt ctgaaaaaaa agaaaattga agatgatatg
      301 agcattagca ttaccgatga gaatagctat ctgctggata atctgaccgg tgatgtgtat
      361 tttgatggtg atgaagccga aatcaccttt cagaaagcaa ttctggaact gccggataaa
      421 cagcgtctgg tttttaatct gcgctattat gatgagatgc cgtatgaaaa aatggcagaa
      481 attaccggca ccagcgaagg tgcactgaaa gcaagctatc accatgcagt taaaaaactg
      541 gaacgttttc tgagtccgga tgattaagga tccaagcttg cggccgcact cgagcaccac
      601 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      661 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      721 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      781 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      841 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      901 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      961 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1021 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1081 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1141 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1201 tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1261 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1321 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1381 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1441 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1501 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1561 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1621 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1681 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1741 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1801 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1861 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     1921 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1981 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2041 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2101 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2161 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2221 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2281 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2341 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2401 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2461 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2521 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2581 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2641 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2701 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2761 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2821 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2881 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2941 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3001 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3061 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3121 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3181 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     3241 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3421 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3481 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3541 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3601 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3661 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3721 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3781 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3841 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3901 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3961 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4021 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4081 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4141 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4201 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4261 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4321 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4501 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4561 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4621 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4681 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4741 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4801 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4861 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4921 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4981 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5041 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5101 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5161 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5221 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5281 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5341 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5401 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5461 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5521 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5581 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5641 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5701 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5761 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5821 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     5881 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5941 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggaag
     6001 ttctgttcca ggggccccat
//
