LOCUS       sequence-176805-        5107 bp    DNA     linear   UNA 15-APR-2025
DEFINITION  PV3 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5107)
  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 5107)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..5107
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     466..488
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     550..569
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    668..701
                     /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(703..736)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      746..755
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             752..1462
                     /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(897..915)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1165..1184
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1648..1667)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1713..1768
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1767..1786)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2129..2145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2129..2145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2142..2164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2153..2169
                     /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(2177..2183,2184..2201,2202..2207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2222..2243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2328..2462
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2482..2616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2519..2538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2573..2592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2684..2701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2855..3443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2935..2954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3614..4405,4406..4474))
                     /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     4237..4256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4475..4579)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4610..4989
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
       61 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
      121 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
      181 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
      241 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
      301 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
      361 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
      421 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
      481 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
      541 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta
      601 attcgagcga aggcgcgccg cactgaaggt cctcaatcgc actggaaaca tcaaggtcgg
      661 cacgcgtgaa gttcctattc tctagaaagt ataggaactt cgataacttc gtatagcata
      721 cattatacga agttataccg gtgccgccac catggtgagc aagggcgagg aggataacat
      781 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
      841 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
      901 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
      961 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1021 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1081 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1141 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     1201 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     1261 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     1321 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     1381 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     1441 catggacgag ctgtacaagt aagaattcgg taccctgacc tcctgccagc aatagtaaga
     1501 caacacgcaa agtcgcggcc gcccaggata catagattac cacaactccg agcccttcca
     1561 ccgctagcgc actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     1621 tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat
     1681 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     1741 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     1801 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     1861 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     1921 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     1981 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     2041 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga
     2101 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2161 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2221 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2281 acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2341 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2401 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2461 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     2521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2581 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     2641 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2701 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2761 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2821 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2881 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2941 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3001 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3061 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3121 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3181 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3241 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3301 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3361 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3421 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3481 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3541 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3601 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3661 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3721 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3781 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3841 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3901 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3961 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4021 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4081 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4141 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4201 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4261 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4321 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4381 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4441 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4501 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4561 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcg acattgatta
     4621 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     4681 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     4741 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     4801 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     4861 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     4921 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     4981 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     5041 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     5101 ggggggg
//
