LOCUS       sequence-74428-e        5996 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 5996)
  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 5996)
  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: pVRa04_1617
FEATURES             Location/Qualifiers
     source          1..5996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             571..588
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(641..659)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      655..702
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      739..1194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(826..845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1036..1057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1221..1325
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1326..1394,1395..2186)
                     /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(1544..1563)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2357..2945
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2846..2865
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3099..3116
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3131..3273
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3199..3218)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3359..3381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3375..3566)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4375..5457)
                     /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     5419..5438
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5458..5535)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5741..5760
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5844..5863
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5844..5862
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5863..5887
                     /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             5902..5924
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5943..5960
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5970..5993
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgcgtgcac gtgaagaact gtttaaaggt ctggttgcag aacatcaaga aatggttgtg
       61 aatacctgct atcgctttgt gtttaatcgt gaagatgccg aagatctggc acaggatgtt
      121 tttgttgaag tttttcgtag cctggaacag tttcgtgaag aagcaaaact gagcacctgg
      181 atttatcgta ttgcagttac caaaagcctg gatcatctgc gtcgtctgaa acgtaaaaaa
      241 cgctttagca gcctgaaacg cattattggt gttagcgatc cgagcgaaga aattgcagca
      301 ccgaccaccg ataatccggc agaagcactg gcaggtagcg aacaggcacg tatcctgcag
      361 aatgccctga atagcctgcc ggatagccag aaaaccgcat ttctgctgag caaacaagaa
      421 ggttatagca atcaagagat tgccgatctg ctgaaaacca gcgttagcgc agttgaaagc
      481 ctggtgcatc gtgcaaaaaa aaacctgcac gataaactgt atgcctatta ttccgaacag
      541 taaggatcca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
      601 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      661 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      721 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      781 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1081 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1141 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttcaggtg
     1201 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1261 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1321 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1381 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1441 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1501 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1561 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1621 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     1681 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1741 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1801 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1861 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1921 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1981 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2041 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2101 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2161 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2221 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2281 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2341 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2401 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2461 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2521 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2581 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2641 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2701 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2761 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2821 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2881 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2941 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3001 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3061 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3121 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3181 tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     3241 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3301 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3361 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3421 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3481 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3541 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3601 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3661 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3721 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3781 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3841 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3901 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3961 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4021 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4081 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     4141 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     4201 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     4261 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     4321 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4381 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4441 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4501 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     4561 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     4621 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     4681 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     4741 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     4801 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4861 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     4921 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4981 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5041 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     5101 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     5161 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     5221 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     5281 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     5341 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     5401 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     5461 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     5521 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     5581 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     5641 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     5701 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5761 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5821 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     5881 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca
     5941 gccatcatca tcatcatcac agcagcggcc tggaagttct gttccagggg ccccat
//
