LOCUS       sequence-74489-e        6026 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 6026)
  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 6026)
  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: pVRa38_1322
FEATURES             Location/Qualifiers
     source          1..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             601..618
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(671..689)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      685..732
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      769..1224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(856..875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1066..1087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1251..1355
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1356..1424,1425..2216)
                     /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(1574..1593)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2387..2975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2876..2895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3129..3146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3161..3303
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3229..3248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3389..3411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3405..3596)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4405..5487)
                     /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     5449..5468
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5488..5565)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5771..5790
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5874..5893
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5874..5892
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5893..5917
                     /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             5932..5954
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5973..5990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6000..6023
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgccggtta ttgcaccgat gcctgcagca cgtccggcac gtattaccaa tcagcgtgat
       61 ggtgcagaag atgcagcagc aggcaccacc gttgatcatc tgaccgaaac ctatcgtgca
      121 cattatcgta gcctgctggg tctggcagca ctgctgctgg atgataccgc aagctgtgaa
      181 gatgttgttc aagaagcatt tatccgcgtt catagcgcac gtaaacgtgt tcgtgatccg
      241 gaaaaaaccc tggcatatct gcgtcagacc gttgttaatc tgagccgtag cgcactgcgt
      301 cgtcgtattc tgggtctgaa actgctgagc aaaccgatgc cggatatggc aagtgccgaa
      361 gaaggtgcat acgatcagct ggaacgtgat agcctgatta aagcaatgaa aggtctgcag
      421 cgtcgtcagc gtgaagttct ggttctgcgt tattttgcag atatgaccga agcacaggtt
      481 gcagaaaccc tgggtattag cctgggtagc gttaaagcct atggtagccg tggtattgca
      541 gccctgcgtg ttgcaatgga agcaccggca taaggatcca agcttgcggc cgcactcgag
      601 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      661 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      721 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      961 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1021 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1081 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1141 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1201 acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1261 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1321 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1381 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1441 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1501 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1561 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1621 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1681 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1741 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1801 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1861 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     1921 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1981 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2041 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     2101 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2161 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2221 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2281 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2341 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2401 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2461 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2521 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2581 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2641 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2701 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2761 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2821 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2881 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2941 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3001 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3061 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3121 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3181 ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc
     3241 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3301 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3361 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3421 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3481 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3541 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3601 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3661 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3721 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3781 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3841 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3901 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3961 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4021 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4081 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4141 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4201 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4261 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4321 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4381 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4441 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4501 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4561 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4621 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4681 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4741 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4801 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4861 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4921 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4981 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5041 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5101 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5161 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5221 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5281 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5341 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5401 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5461 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5521 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5581 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5641 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5701 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     5761 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     5821 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     5881 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5941 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc
     6001 tggaagttct gttccagggg ccccat
//
