LOCUS       sequence-242525-        5439 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  to express Cas9 from S. pyogenes with 2A-EGFP and sgRNA targeting 
            human SCGB3A2 locus at the end of the endogenous coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5439)
  AUTHORS   McCauley KB, Alysandratos KD, Jacob A, Hawkins F, Caballero IS, 
            Vedaie M, Yang W, Slovik KJ, Morley M, Carraro G, Kook S, Guttentag 
            SH, Stripp BR, Morrisey EE, Kotton DN
  TITLE     Single-Cell Transcriptomic Profiling of Pluripotent Stem 
            Cell-Derived SCGB3A2+ Airway Epithelium.
  JOURNAL   Stem Cell Reports. 2018 May 8;10(5):1579-1595. doi: 
            10.1016/j.stemcr.2018.03.013. Epub 2018 Apr 12.
  PUBMED    29657097
REFERENCE   2  (bases 1 to 5439)
  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.2018.03.013"; journalName: "Stem Cell Reports"; 
            date: "2018-05-8- 8"; volume: "10"; issue: "5"; pages: "1579-1595"
COMMENT     Sequence Label: pU6-SCGB3A2_gRNA1-SpCas9-T2A-GFP
FEATURES             Location/Qualifiers
     source          1..5439
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..69
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             76..96
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             121..4221
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             121..4221
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4222..4269
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4285..4338
                     /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             join(4339..4341,4342..5052)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4339..4341,4342..5052)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4642..4661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4989..5010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5080..5097)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5086..5293
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5302..5431
                     /label=AAV2 ITR
ORIGIN
        1 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
       61 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
      121 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
      181 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
      241 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
      301 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
      361 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
      421 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
      481 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
      541 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
      601 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
      661 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
      721 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
      781 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
      841 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
      901 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
      961 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     1021 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     1081 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     1141 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     1201 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     1261 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     1321 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     1381 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     1441 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     1501 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     1561 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     1621 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     1681 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     1741 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     1801 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     1861 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     1921 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     1981 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     2041 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     2101 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     2161 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     2221 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     2281 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     2341 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     2401 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     2461 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     2521 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     2581 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     2641 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     2701 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     2761 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     2821 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     2881 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     2941 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     3001 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     3061 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     3121 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     3181 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     3241 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     3301 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     3361 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     3421 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     3481 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     3541 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     3601 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     3661 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     3721 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     3781 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     3841 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     3901 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     3961 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     4021 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     4081 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     4141 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     4201 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     4261 aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc
     4321 gaggagaatc ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4381 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4441 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4501 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     4561 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4621 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4681 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4741 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4801 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4861 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4921 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4981 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     5041 gagctgtaca aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt
     5101 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     5161 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     5221 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     5281 ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     5341 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     5401 gggcggcctc agtgagcgag cgagcgcgca gctgcctgc
//
