LOCUS       sequence-173422-        3616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PV4 for 2-input, 2-output circuit library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3616)
  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 3616)
  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..3616
                     /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
     promoter        1017..1265
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1017..1037
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1188..1207
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1322..1355)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_RNA        1384..1459
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1651..1670)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1716..1771
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1770..1789)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2132..2148)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2132..2148)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2145..2167)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2156..2172
                     /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(2180..2186,2187..2204,2205..2210))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2225..2246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      2331..2465
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2485..2619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2522..2541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2576..2595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2687..2704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2858..3446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2938..2957)
                     /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 actagtgagg
     1021 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1081 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1141 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1201 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1261 acaccggcgc gccctgacct cctgccagca atagtaagac aacacgcaaa gtcgcacgcg
     1321 tataacttcg tatagcatac attatacgaa gttataccgg tcaccgggtc ttcgagaaga
     1381 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     1441 agtggcaccg agtcggtgct tttttgaatt cggtaccgag ccaactccct ttacaacctc
     1501 actcaagtcc gttagaggcg gccgcccagg atacatagat taccacaact ccgagccctt
     1561 ccaccgctag cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     1621 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     1681 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     1741 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     1801 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     1861 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     1921 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     1981 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     2041 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     2101 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2161 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2221 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2281 gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct
     2341 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2401 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     2461 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     2521 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2581 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     2641 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2701 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2761 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2821 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2881 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2941 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3001 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3061 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3121 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3181 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3241 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3301 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3361 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3421 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3481 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3541 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3601 aaacttggtc tgacag
//
