LOCUS       sequence-74468-e        5954 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 5954)
  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 5954)
  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: pVRa30_35
FEATURES             Location/Qualifiers
     source          1..5954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             529..546
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(599..617)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      613..660
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      697..1152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(784..803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     994..1015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1179..1283
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1284..1352,1353..2144)
                     /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(1502..1521)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2315..2903
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2804..2823
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3057..3074
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3089..3231
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3157..3176)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3317..3339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3333..3524)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4333..5415)
                     /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     5377..5396
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5416..5493)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5699..5718
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5802..5821
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5802..5820
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5821..5845
                     /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             5860..5882
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5901..5918
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5928..5951
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgaaaaaaa aacagaccac caaagcactg ctggttacct gtattaccga tcataaacag
       61 gatttttatc gcctggcctt cagctatgtg aaaaatcagg atgatgccct ggatattgtg
      121 caagaaagca ttaaaaaagc cctgagcagc gttgaaaccg ttcgtaatcc ggaaaccatt
      181 aaaagctggt tctacaaaat tctggtgcgt accgcaattg attttctgcg taaacagaaa
      241 aaaattcgcg tgatggatga cgaaaccatt gaatttctga gcaaaggcaa agaggaccac
      301 tataaagata ccgatctgca tgaagcactg gatgaactgc cgtatcgtta taaaaccatt
      361 atcatcctgc gcttttttga ggatctgaaa ctggaagaaa tcgcagagat taccggtgaa
      421 aataccaata ccgttaaaac ccgtctgtat cgtgcactga aactgatgcg tattcagctg
      481 accaaagaag atctgagtta aggatccaag cttgcggccg cactcgagca ccaccaccac
      541 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      601 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      661 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      721 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      781 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      841 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      901 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      961 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1021 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1081 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1141 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1201 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1261 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1321 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1381 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1441 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1501 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     1561 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1621 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1681 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1741 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1801 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     1861 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1921 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     1981 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2041 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2101 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2161 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2221 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2281 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2341 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2401 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2461 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2521 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2581 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2641 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2701 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2761 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2821 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2881 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2941 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3001 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3061 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3121 gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata
     3181 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3241 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3301 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3361 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3421 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3481 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3541 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3601 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3661 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3721 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3781 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3841 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3901 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3961 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4021 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4081 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4141 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4201 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4261 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4321 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4441 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4501 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4561 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4621 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4681 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4741 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4801 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4861 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4921 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4981 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5041 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5101 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5161 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5221 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5281 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5341 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5401 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5461 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5521 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5581 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5641 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5701 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5761 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     5821 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     5881 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gaagttctgt
     5941 tccaggggcc ccat
//
