LOCUS       sequence-173402-        6179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PV1 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6179)
  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 6179)
  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: pBW705_pCAG-PV1-FRT-lox2272-STOP
FEATURES             Location/Qualifiers
     source          1..6179
                     /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=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)."
     CDS             2912..3706
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2966..2985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3576..3595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3886..4020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4214..4233)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4279..4334
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4333..4352)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4695..4711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4695..4711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4708..4730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4719..4735
                     /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(4743..4749,4750..4767,4768..4773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4788..4809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4894..5028
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5048..5182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5250..5267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5421..6009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5501..5520)
                     /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 cgcgcccatt actcgcatcc
     2761 attctcaggc tgtctcgtct cgtctcgcac gcgtgaagtt cctattctct agaaagtata
     2821 ggaacttcga taacttcgta taggatactt tatacgaagt tataccggtc ggatttgatc
     2881 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2941 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3001 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3061 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3121 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3181 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3241 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3301 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3361 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3421 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3481 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3541 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3601 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3661 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3721 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3781 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3841 cagcgcgggg atctcatgct ggagttcttc gcccacccca tcgataactt gtttattgca
     3901 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3961 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     4021 aaatccgaga attcggtacc gctgggagtt cgtagacgga aacaaacgca gaatccaagc
     4081 gcggccgccc aggatacata gattaccaca actccgagcc cttccaccgc tagcgcactc
     4141 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     4201 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     4261 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     4321 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     4381 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     4441 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     4501 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     4561 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     4621 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc
     4681 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4741 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4801 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4861 cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccagttccg
     4921 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4981 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5041 aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5101 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5161 tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc
     5221 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5281 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5341 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5401 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5461 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5521 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5581 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5641 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5701 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5761 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5821 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5881 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5941 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6001 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6061 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6121 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacag
//
