LOCUS       sequence-177159-        5889 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PV4 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5889)
  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 5889)
  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: pBW708_pCAG-PV4-loxP-GFP-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..5889
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     470..492
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     554..573
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(672..705)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      735..744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(741..743,744..746,747..1457)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(741..743,744..746,747..1457)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(786..807)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1047..1066)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1394..1415
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1458..1511
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1512..2222
                     /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(1657..1675)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1925..1944
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2428..2447)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2493..2548
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2547..2566)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2909..2925)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2909..2925)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2922..2944)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2933..2949
                     /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(2957..2963,2964..2981,2982..2987))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3002..3023
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      3108..3242
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3262..3396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3299..3318)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3353..3372
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3464..3481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3635..4223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3715..3734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4394..5185,5186..5254))
                     /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     5017..5036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5255..5359)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5390..5769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
       61 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
      121 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
      181 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
      241 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
      301 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
      361 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
      421 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
      481 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
      541 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg
      601 attaattcga gcgaaggcgc gccctgacct cctgccagca atagtaagac aacacgcaaa
      661 gtcgcacgcg tataacttcg tatagcatac attatacgaa gttataccgg tagtctcgca
      721 gcaacgctcg tgccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      781 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      841 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      901 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      961 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1021 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1081 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1141 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1201 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1261 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1321 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1381 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1441 tggacgagct gtacaaggag ggcaggggaa gtcttctaac atgcggggac gtggaggaaa
     1501 atcccggccc aatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1561 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1621 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1681 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1741 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1801 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1861 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1921 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1981 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2041 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2101 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2161 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2221 aagcactagc gtcaggtacg ctgaattcgg taccgagcca actcccttta caacctcact
     2281 caagtccgtt agaggcggcc gcccaggata catagattac cacaactccg agcccttcca
     2341 ccgctagcgc actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     2401 tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat
     2461 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     2521 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     2581 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     2641 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     2701 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     2761 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     2821 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga
     2881 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2941 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3001 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3061 acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3121 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3181 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3241 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     3301 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3361 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     3421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcg acattgatta
     5401 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     5461 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     5521 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     5581 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     5641 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     5701 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     5761 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     5821 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     5881 ggggggggg
//
