LOCUS       sequence-74494-e        6329 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 6329)
  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 6329)
  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: pVRa41_491
FEATURES             Location/Qualifiers
     source          1..6329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             904..921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(974..992)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      988..1035
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1072..1527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1159..1178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1369..1390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1554..1658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1659..1727,1728..2519)
                     /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(1877..1896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2690..3278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3179..3198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3432..3449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3464..3606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3692..3714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3708..3899)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4708..5790)
                     /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     5752..5771
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5791..5868)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6074..6093
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6177..6196
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6177..6195
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6196..6220
                     /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             6235..6257
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6276..6293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6303..6326
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgaccgttg cagaaaccat ttttgaaacc cgtcgtcgtg caatgaccgg tctggcatat
       61 cgtatgctgg gtagccgtgc cgaagccgaa gatgttgttc aggatgcatg gctgcgttgg
      121 cgtggtgttg atgttagcga agttcaagaa ccggcagcat ttctgagccg tgttgttacc
      181 cgtctggcac tggatcgtct gaaaagcgca cgtgcccgtc gtgaagttta tgttggtgaa
      241 tggctgccgg aaccggttgt tgatccggat tttgatgcag gtcgtgatgc agatctgagc
      301 gttgcatttc tgctggccct ggaacgcctg acaccgctgg aacgtgcagc ctttctgctg
      361 catgatgttt ttgatacccc gtttgcagaa attgcagcaa ccctgggtcg tagcgaaacc
      421 gcatgtcgtc gtctggcaag ccgtgcacgt gaacatgtgc gtcgtgatcg tccgcgtcat
      481 cagccgagcg ttgatcaaga acgtcgtctg accgcagcat tttttgaagc agttatgacc
      541 ggtgatgaag cagcactgcg tgatctgctg gcacaggatg ttgttatgca tgcagatggt
      601 ggtggtctgg ccagcgcaaa tctgaatccg gttcgtggcc tggcaaaagc agttgcactg
      661 attctgggtg ttcgtcgtaa atggcctccg cctagcgcaa ccgttgttcg cctggcacgt
      721 gttaatggtc agcctggttt tgttctggca gttgatggta gcgtttttca gaccgttggt
      781 ctggaagtgg atgccggtcg tattgttgca gtttataccg ttcgtaatcc ggaaaaactg
      841 gcacatctgg acccgcaggc actgggtctg ggttaaggat ccaagcttgc ggccgcactc
      901 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      961 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1021 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1081 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1141 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1201 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1261 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1321 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1381 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1441 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1501 ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     1561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1621 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1681 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1741 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1801 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1861 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1921 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1981 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2041 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2101 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2161 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     2221 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2281 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2341 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2401 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2461 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2521 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2581 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2821 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3181 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3241 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3301 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3361 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3421 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3481 tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3601 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3721 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3781 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3841 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3901 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3961 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4021 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4081 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4141 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4201 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4261 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4321 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4381 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4441 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4501 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4561 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4621 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4681 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4801 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4861 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4921 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4981 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5041 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5101 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5161 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5221 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5281 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5341 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5401 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5461 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5521 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5581 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5641 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5701 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5761 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5821 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5881 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5941 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6001 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6061 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     6181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     6241 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
     6301 gcctggaagt tctgttccag gggccccat
//
