LOCUS       sequence-274492-        4465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Library reporter vector/integration vector. Contains library cloning
            sites MluI and SpeI upstream of a terminator and downstream of a 
            BxB1 attB site. Library integration occurs through BxB1 site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4465)
  AUTHORS   Davis JE, Insigne KD, Jones EM, Hastings QA, Boldridge WC, Kosuri S
  TITLE     Dissection of c-AMP Response Element Architecture by Using Genomic 
            and Episomal Massively Parallel Reporter Assays.
  JOURNAL   Cell Syst. 2020 Jul 22;11(1):75-85.e7. doi: 
            10.1016/j.cels.2020.05.011. Epub 2020 Jun 29.
  PUBMED    32603702
REFERENCE   2  (bases 1 to 4465)
  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"; doi: 
            "10.1016/j.cels.2020.05.011"; journalName: "Cell Syst"; date: 
            "2020-07-22- 22"; volume: "11"; issue: "1"; pages: "75-85.e7"
COMMENT     Sequence Label: Donor_eGP2AP_RC
FEATURES             Location/Qualifiers
     source          1..4465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     665..684
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(758..862)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     930..948
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(986..1008)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1108..1127
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    1158..1382
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1521..1558
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(1569..1571,1572..1574,1575..2285)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1569..1571,1572..1574,1575..2285)
                     /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(1614..1635)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1875..1894)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2222..2243
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2295..2351
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2352..2948
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2845..2865
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2955..3179
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3188..3221
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     primer_bind     complement(3246..3262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3246..3262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3259..3281)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3270..3286
                     /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(3294..3300,3301..3318,3319..3324))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3339..3360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3477..3494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3648..4236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3728..3747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4407..4465,1..757))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(4407..4465,1..757))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
       61 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      121 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      181 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      241 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
      301 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
      361 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
      421 attcttctaa tacctggaat gctgttttcc cagggatcgc agtggtgagt aaccatgcat
      481 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
      541 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
      601 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
      661 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
      721 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt
      781 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
      841 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
      901 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
      961 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1021 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1081 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcaca
     1141 agcttgcatg cctgcagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     1201 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     1261 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     1321 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     1381 agtcgaccgg tgactgacgt aaccaccctg atcgacgggg agtgtactag tatgaggtca
     1441 caagtgtgac tgaaacgcgt gctctacgac tatgctcttc ccaactgcta gccgtatcat
     1501 cgagctcacg tgaagcttcc ggcttgtcga cgacggcggt ctccgtcgtc aggatcatcc
     1561 gggaattcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1621 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1681 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1741 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1801 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1861 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1921 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1981 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2041 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2101 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2161 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2221 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2281 acaagggaag cggagcgacg aattttagtc tactgaaaca agcgggagac gtggaggaaa
     2341 accctggacc taccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     2401 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2461 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2521 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2581 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2641 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2701 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2761 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2821 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcactg
     2881 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     2941 gtgcctgacc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3001 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3061 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3121 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggt
     3181 taattaaata acttcgtata atgtatacta tacgaagtta tggcgcgccg ttaacgatat
     3241 ccatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3301 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3361 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3421 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3481 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3541 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3601 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3661 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3721 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3781 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3841 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3901 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3961 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4021 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4081 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4141 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4201 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4261 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4321 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4381 atatatgagt aaacttggtc tgacagttag aaaaactcat cgagcatcaa atgaaactgc
     4441 aatttattca tatcaggatt atcaa
//
