LOCUS       sequence-177010-        6171 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PV8 for 3-input, 2-output Full Adder.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6171)
  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 6171)
  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: pBW811_pCAG-PV8-F14-GFP-T2A-mCherry-BGHpA
FEATURES             Location/Qualifiers
     source          1..6171
                     /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"
     regulatory      731..740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(737..739,740..742,743..1453)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(737..739,740..742,743..1453)
                     /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(782..803)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1043..1062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1390..1411
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1454..1507
                     /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             1508..2218
                     /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(1653..1671)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1921..1940
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2239..2463
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2709..2728)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2774..2829
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2828..2847)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3190..3206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3190..3206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3203..3225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3214..3230
                     /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(3238..3244,3245..3262,3263..3268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3283..3304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      3389..3523
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3543..3677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3580..3599)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3634..3653
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3745..3762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3916..4504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3996..4015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4675..5466,5467..5535))
                     /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     5298..5317
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5536..5640)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5671..6050
                     /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 aggcgcgccc ctcgtctcaa ccaaagcaat caacccatca accacctggg
      661 cacgcgtgaa gttcctattc tatcagaagt ataggaactt caccggtagt ctcgcagcaa
      721 cgctcgtgcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      781 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      841 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      901 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      961 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1021 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1081 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1141 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1201 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1261 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1321 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1381 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1441 cgagctgtac aaggagggca ggggaagtct tctaacatgc ggggacgtgg aggaaaatcc
     1501 cggcccaatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     1561 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     1621 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     1681 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     1741 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     1801 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     1861 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     1921 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     1981 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     2041 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     2101 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     2161 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaagc
     2221 actagcgtca ggtacgctct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2281 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2341 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2401 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2461 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2521 gcggaattcg gtaccgttcc ttatcatctg gcgaatcgga cccacaagag cactggcggc
     2581 cgcccaggat acatagatta ccacaactcc gagcccttcc accgctagcg cactcctcag
     2641 gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac
     2701 cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat
     2761 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     2821 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     2881 ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta
     2941 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     3001 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     3061 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     3121 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc
     3181 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3241 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3301 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     3361 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccag ttccgcccat
     3421 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     3481 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3541 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3601 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3661 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3721 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3781 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3841 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3901 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3961 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4021 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4081 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4141 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4201 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4261 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4321 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4381 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4441 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4501 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4561 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4621 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4681 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4741 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4801 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4861 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4921 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4981 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5041 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5101 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5161 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5221 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5281 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5341 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5401 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5461 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5521 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5581 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5641 cacatttccc cgaaaagtgc cacctgggtc gacattgatt attgactagt tattaatagt
     5701 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     5761 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     5821 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     5881 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     5941 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     6001 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
     6061 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt
     6121 tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg g
//
