LOCUS       sequence-173404-        5740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PV3 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5740)
  AUTHORS   Weinberg BH, Pham NT, Caraballo LD, Lozanoski T, Engel A, Bhatia S, 
            Wong WW
  TITLE     Large-scale design of robust genetic circuits with multiple inputs 
            and outputs for mammalian cells.
  JOURNAL   Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805.
  PUBMED    28346402
REFERENCE   2  (bases 1 to 5740)
  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: "Nat 
            Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3805."
COMMENT     Sequence Label: pBW707_pCAG-PV3-FRT-loxP-mCherry
FEATURES             Location/Qualifiers
     source          1..5740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        997..1376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1378..1653
                     /label=chicken beta-actin promoter
     intron          1654..2669
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2593..2615
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2677..2696
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2795..2828
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(2830..2863)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2873..2882
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2879..3589
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3024..3042)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3292..3311
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3775..3794)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3840..3895
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3894..3913)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4256..4272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4256..4272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4269..4291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4280..4296
                     /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)."
     promoter        complement(join(4304..4310,4311..4328,4329..4334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4349..4370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4455..4589
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4609..4743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4646..4665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4700..4719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4811..4828)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4982..5570)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5062..5081)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
     1021 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1081 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1141 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1201 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1261 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1321 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1381 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1441 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
     1501 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggaa aggtgcggcg
     1561 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1621 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgttgcc ttcgccccgt
     1681 gccccgctcc gcgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1741 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
     1801 cggctcgttt cttttctgtg gctgcgtgaa agccttaaag ggctccggga gggccctttg
     1861 tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg
     1921 cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     1981 ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg
     2041 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     2101 cgcggcggtc gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc
     2161 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     2221 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     2281 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     2341 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     2401 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     2461 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     2521 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     2581 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     2641 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     2701 ctcatcattt tggcaaagaa ttgattaatt cgagcgaagg cgcgccgcac tgaaggtcct
     2761 caatcgcact ggaaacatca aggtcggcac gcgtgaagtt cctattctct agaaagtata
     2821 ggaacttcga taacttcgta tagcatacat tatacgaagt tataccggtg ccgccaccat
     2881 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     2941 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     3001 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     3061 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     3121 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     3181 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     3241 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     3301 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     3361 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     3421 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     3481 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     3541 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag aattcggtac
     3601 cctgacctcc tgccagcaat agtaagacaa cacgcaaagt cgcggccgcc caggatacat
     3661 agattaccac aactccgagc ccttccaccg ctagcgcact cctcaggtgc aggctgccta
     3721 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt
     3781 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     3841 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     3901 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     3961 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca
     4021 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
     4081 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc
     4141 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
     4201 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat
     4261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     4441 gtcagcaacc atagtcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4501 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4561 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt
     4621 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4681 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4741 atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4801 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4861 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4921 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4981 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5041 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5101 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5161 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5221 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5281 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5341 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5401 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5461 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5521 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5581 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5641 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5701 aatcaatcta aagtatatat gagtaaactt ggtctgacag
//
