LOCUS       sequence-74441-e        6059 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 6059)
  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 6059)
  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_524
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             634..651
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(704..722)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      718..765
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      802..1257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(889..908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1099..1120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1284..1388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1389..1457,1458..2249)
                     /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(1607..1626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2420..3008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2909..2928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3162..3179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3194..3336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3262..3281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3422..3444
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3438..3629)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4438..5520)
                     /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     5482..5501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5521..5598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5804..5823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5907..5926
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5907..5925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5926..5950
                     /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             5965..5987
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6006..6023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6033..6056
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggttgcag aacaggttgg tcgtagtccg cctgatccgg ttcgtgataa ttcatttaaa
       61 cgtgaactgg ttaccctgat tccgcatctg cgtgcatttg cacgtaccct gaccggtgat
      121 ccgaccgcag cagatgatct ggcacaggat gcaatgatga aagcatggga tgcacgtgcc
      181 agctttcagc tgggcaccaa tatgaaagcc tggaccttta tgattctgcg caatcagttc
      241 tatagcgaaa aacgtcgtag ctggcgtcag agccaactgg atcaagaagc agcagaacgc
      301 accctggttg cagttgatga tccggaagca ccggttgcac tggatgaact gcgtcagggt
      361 ctgagcatgc tgcctgccga acagcgtgaa gcactgattc tggttggtgc cggtggtttt
      421 gcctatgaag aagccgctga aatttgtaat tgtgcagttg gcaccgttaa aagccgtgtt
      481 agccgtgcac gtcgtgccct gcaggcaatt ctggaaagcg gtgcctatga tcgtgatggt
      541 gcagcagccg gtgatgcaat gcgtgccatt ctggcagatg cagaacgtct gagcgcactg
      601 cgttaaggat ccaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
      661 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
      721 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
      781 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
      841 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      901 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      961 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1021 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1081 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1141 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1201 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
     1261 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1321 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1381 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1441 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1501 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1561 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1621 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     1681 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1741 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1801 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     1861 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     1921 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     1981 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2041 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2101 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2161 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2221 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2281 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     2341 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2401 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2461 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2521 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2581 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2641 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2701 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2761 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2821 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2881 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2941 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3001 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3061 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3121 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3181 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3241 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3301 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3361 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3421 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3481 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3541 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3601 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3661 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3721 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3781 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3841 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3901 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3961 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4021 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4081 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4141 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4201 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4261 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4321 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4381 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4441 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4501 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4561 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4621 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4681 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4741 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4801 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4861 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4921 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4981 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5041 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5101 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5161 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5221 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5281 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5341 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5401 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5461 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5521 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5581 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5641 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5701 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5761 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5821 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5881 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     5941 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
     6001 gcagccatca tcatcatcat cacagcagcg gcctggaagt tctgttccag gggccccat
//
