LOCUS       sequence-74450-e        5987 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 5987)
  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 5987)
  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: pVRa17_1691
FEATURES             Location/Qualifiers
     source          1..5987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             562..579
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(632..650)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      646..693
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      730..1185
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(817..836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1027..1048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1212..1316
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1317..1385,1386..2177)
                     /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(1535..1554)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2348..2936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2837..2856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3090..3107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3122..3264
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3190..3209)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3350..3372
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3366..3557)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4366..5448)
                     /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     5410..5429
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5449..5526)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5732..5751
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5835..5854
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5835..5853
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5854..5878
                     /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             5893..5915
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5934..5951
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5961..5984
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggcacgtg ttagcggtgc agcagcagcc gaagcagcac tgatgcgtgc actgtatgat
       61 gaacatgcag cagttctgtg gcgttatgca ctgcgtctga ccggtgatgc agcacaggca
      121 gaagatgttg ttcaagaaac cctgctgcgt gcatggcagc atccggaagt tattggtgat
      181 accgcacgtc cggcacgcgc atggctgttt accgttgcac gtaatatgat tattgatgaa
      241 cgtcgtagcg cacgttttcg taatgttgtt ggtagcaccg atcagagcgg tacaccggaa
      301 cagagcacac cggatgaagt taatgcagca ctggatcgtc tgctgattgc agatgcactg
      361 gcacagctga gcgcagaaca tcgtgcagtt attcagcgta gctattatcg tggttggagc
      421 accgcacaga ttgcaaccga tctgggtatt gcagaaggca ccgttaaaag ccgtctgcat
      481 tatgcagttc gtgccctgcg cctgaccctg caagaactgg gtgttacccg ttaaggatcc
      541 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
      601 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
      661 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
      721 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
      781 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      841 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      901 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      961 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1021 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1081 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1141 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc
     1201 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1261 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1321 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1381 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1441 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1501 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1561 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1621 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1681 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1741 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1801 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1861 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1921 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1981 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2041 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2101 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2161 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2221 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2281 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2341 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2401 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2461 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2521 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2581 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2641 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2701 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2761 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2821 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2881 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2941 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3001 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3061 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3121 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     3181 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3241 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3301 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3361 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3421 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3481 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3541 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3601 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3661 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3721 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3781 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3841 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3901 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3961 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4021 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4081 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4141 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4201 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4261 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4321 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4381 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4441 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4501 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4561 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4621 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4681 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4741 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4801 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4861 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4921 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     4981 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5041 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5101 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5161 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5221 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5281 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5341 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5401 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5461 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5521 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5581 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5641 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5701 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     5761 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     5821 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     5881 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc
     5941 atcatcatca cagcagcggc ctggaagttc tgttccaggg gccccat
//
