LOCUS       sequence-74440-e        6038 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 6038)
  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 6038)
  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: pVRa15_436
FEATURES             Location/Qualifiers
     source          1..6038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             613..630
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(683..701)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      697..744
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      781..1236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(868..887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1078..1099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1263..1367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1368..1436,1437..2228)
                     /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(1586..1605)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2399..2987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2888..2907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3141..3158
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3173..3315
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3241..3260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3401..3423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3417..3608)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4417..5499)
                     /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     5461..5480
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5500..5577)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5783..5802
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5886..5905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5886..5904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5905..5929
                     /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             5944..5966
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5985..6002
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6012..6035
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgacccaga ccccgaaagc accggcaaaa catcgtgatc cgcgtgatga actgccggaa
       61 catctgcctg cactgcgtgc atttgcaatt agcctgaccc gtaatgttgc agttgcagat
      121 gatctggttc aggataccat tgttaaagcc tggaccaact tcgataaatt tacggaaggc
      181 accaatctgc gtgcctggct gtttaccatt ctgcgtaata ccttctatag cgataaacgt
      241 aaacatcgtc gtgaagttcc agatccggaa ggtgttcatg cagcaagcct gtttgttaaa
      301 ccggcacatg atggtcatct ggcatttagc gattttagcg cagcatttga tcagctgagc
      361 cctgaacacc gtgaagtgct gattctggtt ggtgcaagcg gttttgccta tgaagatgca
      421 gcacagatga tgggtgttgc cgttggcacc gttaaaagcc gtgcaaatcg tgcacgtgcc
      481 cgtctggcag aactgctggg tctggaaaaa ggtgaagaaa tttttagcgg tgttgatggt
      541 cagaccctgg cagttatgag ccgtagcggt atgaccgcag cataaggatc caagcttgcg
      601 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      661 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      721 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
      781 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      841 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      901 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      961 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1021 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1081 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1141 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1201 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     1261 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1381 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1441 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1501 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1561 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1621 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1681 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1741 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1801 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1861 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     1921 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1981 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2041 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2101 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2161 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2221 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2281 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2341 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2401 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2461 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2521 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2581 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2641 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2701 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2761 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2821 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2881 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2941 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3001 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3061 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3181 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag
     3241 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3301 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3361 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3421 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3481 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3541 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3601 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3661 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3721 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     3781 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     3841 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3901 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     3961 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4021 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4081 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4141 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4201 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4261 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4321 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4381 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4501 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4561 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4621 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4681 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4741 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4801 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4861 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4921 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4981 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5041 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5101 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5161 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5221 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5281 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5341 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5401 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5461 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5521 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5581 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5641 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5701 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5761 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5821 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5881 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     5941 aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc
     6001 acagcagcgg cctggaagtt ctgttccagg ggccccat
//
