LOCUS       sequence-176801-        5084 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PV2 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5084)
  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 5084)
  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: pBW706_pCAG-PV2-lox2272-GFP
FEATURES             Location/Qualifiers
     source          1..5084
                     /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    complement(668..701)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      711..720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(717..719,720..722,723..1436)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(717..719,720..722,723..1433)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(762..783)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1023..1042)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1370..1391
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1622..1641)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1687..1742
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1741..1760)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2103..2119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2103..2119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2116..2138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2127..2143
                     /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(2151..2157,2158..2175,2176..2181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2196..2217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2302..2436
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2456..2590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2493..2512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2547..2566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2658..2675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2829..3417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2909..2928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3588..4379,4380..4448))
                     /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     4211..4230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4449..4553)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4584..4963
                     /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 ctgggagttc gtagacggaa acaaacgcag aatccaagcg
      661 cacgcgtata acttcgtata ggatacttta tacgaagtta taccggtgcc gccaccatgg
      721 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      781 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      841 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      901 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      961 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1021 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1081 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1141 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1201 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1261 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1321 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1381 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagaat
     1441 tcggtaccgc actgaaggtc ctcaatcgca ctggaaacat caaggtcggc ggccgcccag
     1501 gatacataga ttaccacaac tccgagccct tccaccgcta gcgcactcct caggtgcagg
     1561 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     1621 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     1681 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     1741 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     1801 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     1861 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     1921 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     1981 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     2041 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     2101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     2281 tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc cattctccgc
     2341 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     2401 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     2461 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2521 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     2581 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2641 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2701 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2761 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2821 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2881 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2941 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3001 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3061 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3121 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3181 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3241 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3301 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3361 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3421 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3481 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4441 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4501 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4561 ccccgaaaag tgccacctgg gtcgacattg attattgact agttattaat agtaatcaat
     4621 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     4681 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     4741 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     4801 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     4861 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     4921 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     4981 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     5041 tttttaatta ttttgtgcag cgatgggggc gggggggggg gggg
//
