LOCUS       sequence-213391-        7087 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in T2A-H2B-eGFP to the c-terminal of mouse 
            Pdgfra coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7087)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 7087)
  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. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: Pdgfra-T2A-H2B-eGFP donor
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(16..35)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     226..247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        410..514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(515..583,584..1375)
                     /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     complement(733..752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1546..2134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2035..2054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2288..2305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2422..2443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2458..2463,2464..2481,2482..2488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2496..2512
                     /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)."
     primer_bind     2501..2523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2520..2536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2520..2536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2555..2575
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2557..2575
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2604..2620
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2605..2621
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4580..4633
                     /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             4640..5017
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5030..5039
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5036..5038,5039..5041,5042..5755)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5036..5038,5039..5041,5042..5752)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5081..5102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5342..5361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5689..5710
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5757..5773)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5757..5773)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6828..6847)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6829..6847)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6857..6874)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6857..6873)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6866..6888)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
       61 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
      601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
      661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
      721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
      781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
      841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
      901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
      961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1681 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     1921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     1981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     2521 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     2581 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcctctg
     2641 agctcttaaa cagccatgga gcaaggaaaa ccttgagaga ggcggggttg ggatatgagg
     2701 tgctccatca acagccttta gccttgcatg cggtgcagaa ctagctgggg aggactttca
     2761 aactgctgat tcacaggcct ctcaccctgt cagtctgtct gtccctcctt cctctggtgg
     2821 gctgccttct acttctgttt tgtgtgacgc atagggaagc cacctcacaa aggggaagga
     2881 gtcgggggtt ctacccacct gcagatgatc gtgaatttca aagactcaga tggacaaggc
     2941 acatggtgac ccaaggcaga atatggaaaa gccagcaaga atctagagaa gggaggagag
     3001 gtggggattt tgggcaggct ggtgatttta gggatagtaa cactaacacc taacactttt
     3061 gttttaaaaa tgtttagtac acagtagcag tgagtacacc tcccaattag tgggtacagg
     3121 ggttgacatt tgccccattc tctttatcca gattggtgtc acataaatca tcatgactac
     3181 agacacacac acacacacac acacacacac acacacacac cttatttctc acccagtgac
     3241 tgaaagctaa gtagaaaaca ccaagataat tcattcctag tattctgcga cactctttcc
     3301 taagaatacg atcattcatg ctgtgtctat catgaccaca cggaagagag tgaactgtta
     3361 aagaaaatta gagttggagg tatagaattc atgttggcca tatctgaggc cataggttca
     3421 catccctgta ctgaaagaaa aaagagaaga agaaaaggaa gccaacaacc ggtctcagat
     3481 tcatttcata cctgggcttt attcaaattc agttgaataa taactgctaa tttattgagc
     3541 tctttatatg tttatagtta tcagattatt atcgtattta atcatctcag agatctttat
     3601 ttttattttt attatgtata ttttactttt gagattatat ttatgtgtgt gtgtacagat
     3661 ctgtacacct aagtatgtga ctgcaaccgg cttagactgt atgatactcg tatgtgtacg
     3721 ttttcggggc tgaccatttg gtattggata gccaatttgg tgtattcgtc cctggggaag
     3781 gccctttttc tggttctcaa ccttccttcg ttatctgtcg ctcttggtct agaaaagagg
     3841 ctgcctgagc tccttgctta cccattaccg tgcctattgg tgccgttctg gtgcaggtct
     3901 tgcgggcaag gcttctatgc cttgtgtaaa tggtaaactg aagacttttc tcttctgggc
     3961 ctttagaccc agaggggcgg ggtgaacaag aggcaggggc ctggcaggaa gtgaaggaag
     4021 tgtgggatcc catccttctg cttcctaagg tttttttttt cccttctctc tcagtacctt
     4081 ttgattggct gttgcttgtt ctctcccctg ataggctgag gagggaaagt agaaactctt
     4141 actcttgtta gccacgccca caaaagaccc ctggcaggaa aggcagcaaa ctgacctgcg
     4201 cagttggccc tgttttggtt ttgtgttggc tctgtgcagc aatggccagt gagtggctgg
     4261 ctggctcagc cgctcactgg ccatgctcgg ccactcatgc ttcaacagga aaggaccttg
     4321 tgcatgagga aagagtcccg ctgcttcagg cagccaccct ggaaatgcag ccccgtaggc
     4381 agtctgtcca tgaccttagc ctttgctttt caatcccatc agctcacaga cttcggaaga
     4441 gagtgccatc gagacaggtt ccagtagttc caccttcatc aagagagagg acgagaccat
     4501 cgaggacatc gacatgatgg atgacattgg tatagattcc tcggacctgg tggaggacag
     4561 cttcctggat atcgaattcg agggcagagg aagtcttcta acatgcggtg acgtggagga
     4621 gaatcccggc cctaccggta tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg
     4681 ctccaagaag gcggtgacta aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg
     4741 caaggagagc tattccatct atgtgtacaa ggttctgaag caggtccacc ctgacaccgg
     4801 catttcgtcc aaggccatgg gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat
     4861 cgcaggtgag gcttcccgcc tggcgcatta caacaagcgc tcgaccatca cctccaggga
     4921 gatccagacg gccgtgcgcc tgctgctgcc tggggagttg gccaagcacg ccgtgtccga
     4981 gggtactaag gccatcacca agtacaccag cgctaaggat ccaccggtcg ccaccatggt
     5041 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     5101 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     5161 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     5221 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     5281 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     5341 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     5401 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     5461 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     5521 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     5581 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     5641 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     5701 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaggatc
     5761 cactagttct agagcggccg ctaactgaca cgctccgggt atcatcttct ccgggagcct
     5821 cctcgggata accttaagaa aaccacttca ttgcaatgca aaagttgaga agaggacttg
     5881 ggtgatgtgg agagagaaag ttcccgaggc cgagggcctt ggtaagcctg tgtggatgac
     5941 tgggatactt ggattgaact ttatccgtgt tgcctcttgc aacgcgtcag tagcatctca
     6001 gcggcgtgtg cggtttggaa atggatggac aagagagtga ctggccacag aagacgggct
     6061 ttgtgtttcc agggcatggg tgagagtccc acacgcgcca acttgtactg cagacaactc
     6121 cagcattgta attatgtaaa taactcttaa ccgaggatgg gtttagatct gtgttaactc
     6181 tcctctccgg acctctgaag agaccactca gtccacccgt gtgcttcctt ccttcttgga
     6241 atccggtacc agctgctgtt gaattttctc atgaagtaca tgcaaaacca ctttgaactt
     6301 tggaaggtac tggaaccgtc gtgttttact gtcttgtaag tgttaaagag gcaaaaagat
     6361 aaaaactaac caaccgtgag tagtaggttt gggtctttta gggcacccaa gaattcctct
     6421 gtatcttgta atctgtactt ataacactac tactgtttaa caagaatgct aaatatgtaa
     6481 tccgtaacat gatttcttta cagagaaagc acaattaaaa caaaaccctt actaagaagg
     6541 tgacaagttt ggcagttttt gacatttata ttaagtaaca cgtttctcaa taaagtataa
     6601 taggtttagc ggattaaagt tagcaaacac tagagagcag agtgtaagta gttatccaga
     6661 aagtcaaacc tttttgtttt gttctaagca gtctccctaa tccaaggcac taggaacagt
     6721 taatgacctc tgaagtactg ggataagcaa agcagaaacc cggtgggtgt cctaagtgtt
     6781 ctcagtagaa aggcgtgcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt
     6841 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     6901 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     6961 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     7021 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     7081 ttgccag
//
