LOCUS       sequence-74439-e        6227 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 6227)
  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 6227)
  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: pVRa14_3200
FEATURES             Location/Qualifiers
     source          1..6227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             802..819
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(872..890)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      886..933
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      970..1425
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1057..1076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1267..1288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1452..1556
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1557..1625,1626..2417)
                     /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(1775..1794)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2588..3176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3077..3096
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3330..3347
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3362..3504
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3430..3449)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3590..3612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3606..3797)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4606..5688)
                     /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     5650..5669
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5689..5766)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5972..5991
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6075..6094
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6075..6093
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6094..6118
                     /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             6133..6155
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6174..6191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6201..6224
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggaactgc tgggtggtcc gcgtgttggt aataccgaaa gccagctgtg tgttgcagat
       61 ggtgatgatc tgccgaccta ttgtagcgca aatagcgaag atctgaacat taccaccatt
      121 acaaccctga gcccgaccag catgagccat ccgcagcagg ttcgtgatga tcagtgggtt
      181 gaaccgagcg atcagctgca gggcaccgca gtttttgatg caaccggtga taaagcaacc
      241 atgccgagct gggatgaact ggttcgtcag catgcagatc gtgtttatcg tctggcatat
      301 cgtctgagcg gtaatcagca tgatgcagag gatctgaccc aagaaacctt tattcgtgtt
      361 tttcgtagcg tgcagaatta tcagcctggc acctttgaag gttggctgca tcgtattacc
      421 acaaacctgt ttctggatat ggttcgtcgt cgtgcacgta ttcgtatgga agcactgccg
      481 gaagattatg atcgtgttcc ggcagatgaa ccgaatccgg aacaaattta tcatgatgca
      541 cgtctgggtc cggatctgca ggcagcactg gcaagcctgc ctccggaatt tcgtgcagca
      601 gttgttctgt gtgatattga aggtctgtcc tatgaagaaa ttggtgcaac cctgggtgtt
      661 aaactgggca ccgttcgtag ccgtattcat cgtggtcgtc aggcactgcg tgattatctg
      721 gcagcacatc ctgaacatgg tgaatgtgca gttcatgtta atccggttcg ttaaggatcc
      781 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
      841 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
      901 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
      961 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1021 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1081 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1141 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1201 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1261 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1321 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1381 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc
     1441 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1501 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1561 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1621 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1681 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1741 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1801 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1861 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1921 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1981 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2041 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2101 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2161 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2221 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2281 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2341 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2401 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2461 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2521 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2581 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2641 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2701 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2761 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2821 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2881 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2941 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3001 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3061 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3121 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3181 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3241 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3301 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3361 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     3421 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3481 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3541 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3601 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3661 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3721 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3781 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3841 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3901 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3961 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4021 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4081 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4141 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4201 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4261 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4321 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4381 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4441 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4501 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4561 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4681 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4741 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4801 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4861 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4921 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4981 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5041 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5101 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5161 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5221 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5281 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5341 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5401 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5461 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5521 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5581 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5641 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5701 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5761 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5821 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5881 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5941 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6001 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6061 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     6121 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc
     6181 atcatcatca cagcagcggc ctggaagttc tgttccaggg gccccat
//
