LOCUS       sequence-204847-        2364 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor scaffold with EGFP positive selection module, and tagBFP 
            negative selection module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2364)
  AUTHORS   Arias-Fuenzalida J, Jarazo J, Qing X, Walter J, Gomez-Giro G, 
            Nickels SL, Zaehres H, Scholer HR, Schwamborn JC
  TITLE     FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's 
            Disease Modeling.
  JOURNAL   Stem Cell Reports. 2017 Nov 14;9(5):1423-1431. doi: 
            10.1016/j.stemcr.2017.08.026. Epub 2017 Oct 5.
  PUBMED    28988985
REFERENCE   2  (bases 1 to 2364)
  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.stemcr.2017.08.026"; journalName: "Stem Cell Reports"; 
            date: "2017-11-14- 14"; volume: "9"; issue: "5"; pages: "1423-1431"
COMMENT     Sequence Label: pDONOR-tagBFP-PSM-EGFP
FEATURES             Location/Qualifiers
     source          1..2364
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /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(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             718..771
                     /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             772..1368
                     /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     1265..1285
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1447..1466)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1512..1567
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1566..1585)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2097..2159
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2210..2230)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2210..2228)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2249..2265)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2249..2265)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2262..2284)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2273..2289
                     /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(2297..2303,2304..2321,2322..2327))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2342..2363
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggag
      721 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc taccgagtac
      781 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
      841 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
      901 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      961 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     1021 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     1081 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     1141 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     1201 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1261 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1321 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca ctcctcaggt
     1381 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     1441 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     1501 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     1561 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     1621 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     1681 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     1741 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     1801 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     1861 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggatc
     1921 cctcgacttt tgttacttta tagaagaaat tttgagtttt tgtttttttt taataaataa
     1981 ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta aatataataa
     2041 aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat aaaacacatg
     2101 cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc tttctagggt
     2161 taacctagta tacgcgtatg cggccgctta attaatccag cttttgttcc ctttagtgag
     2221 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2281 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2341 aatgagtgag ctaactcaca ttaa
//
