LOCUS       sequence-213390-        6445 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for knock-in T2A-H2B-eGFP to the c-terminal of mouse 
            Gata3 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6445)
  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 6445)
  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: Gata3-T2A-H2B-eGFP donor
FEATURES             Location/Qualifiers
     source          1..6445
                     /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             3860..3913
                     /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             3920..4297
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      4310..4319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4316..4318,4319..4321,4322..5035)
                     /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(4316..4318,4319..4321,4322..5032)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4361..4382)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4622..4641)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4969..4990
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6186..6205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6187..6205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6215..6232)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6215..6231)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6224..6246)
                     /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 gataagcttc cagcatgtcc
     2641 tacaaatctc cagagtaggg ctttgcatct ttgcctgatg cattcatgtt tttttttttt
     2701 tcttgtgacc gtgagtatcc ttacaagcca tggaaggact ttgggtcctg ttggctgttg
     2761 ttaaatcctt gttgattgga tggcttgagt tgcttgtgca gaagcaggaa ggggaaagca
     2821 acagccttga agcactgcag ctgtgttggg tgatgtgggc acagggttat cctttggaag
     2881 agcatactgc tttagcacag gctccggagc tgtggatgtc gctccctggc ttccacccct
     2941 aggaccctat tccctccaaa aatctcagaa cacacacatt tccaggggaa gtttccagaa
     3001 catcaccctg tgaaaaggcc aggtagaaga gtgacagcca gaccttgcag gaaggcacag
     3061 agacccagtt ttctagaaag gtggatttca acacaggagg cttcactctg tctggctctg
     3121 tgtggcaagt cccagagaag actcatcata tacgtaaaca atggtgtctg gcgaccctga
     3181 ttccccatct tctcatgaaa tatcttaggg aaatcttaag gaagtaacac aggttgagga
     3241 agtaacacag gtttttgtat tttagtcatc agatatatac tcgatactag gtgggccccg
     3301 aacttcccct gttgatgagt ttagctgaat tgtgctcggg gcctgtgggc tttgaaagca
     3361 atggcccctg aatcagaagc cctgtgttcc aaggcctgca gtacctgggt ctctggaaag
     3421 aaggaaggag taggtaggaa gaagaggcaa tcaacacagg aacacatccc tggcctgggg
     3481 cctgtgctac tgtgcaaaag cttttatcat ttcaaaggca gcaaaaaagt aaaaaaaaaa
     3541 aaaattgatc tttgtttaga ttaacagacc cctgactatg aagaaagaag gcatccagac
     3601 ccgaaaccgg aagatgtcta gcaaatcgaa aaagtgcaaa aaggtgcatg acgcgctgga
     3661 ggacttcccc aagagcagct ccttcaaccc ggccgctctc tccagacaca tgtcatccct
     3721 gagccacatc tctcccttca gccactccag ccacatgctg accacaccga cgcccatgca
     3781 tccgccctcc ggcctctcct tcggacctca ccacccttcc agcatggtca ccgccatggg
     3841 taagcttgat atcgaattcg agggcagagg aagtcttcta acatgcggtg acgtggagga
     3901 gaatcccggc cctaccggta tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg
     3961 ctccaagaag gcggtgacta aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg
     4021 caaggagagc tattccatct atgtgtacaa ggttctgaag caggtccacc ctgacaccgg
     4081 catttcgtcc aaggccatgg gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat
     4141 cgcaggtgag gcttcccgcc tggcgcatta caacaagcgc tcgaccatca cctccaggga
     4201 gatccagacg gccgtgcgcc tgctgctgcc tggggagttg gccaagcacg ccgtgtccga
     4261 gggtactaag gccatcacca agtacaccag cgctaaggat ccaccggtcg ccaccatggt
     4321 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     4381 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     4441 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     4501 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     4561 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     4621 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     4681 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     4741 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     4801 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     4861 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     4921 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     4981 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaactag
     5041 ttctagagcg gccgcagagg cagagccctg ctccacatgc gtgaggagtc tccaagtgtg
     5101 cgaagagttc ctccgacccc ttctacttgc gtttttcgca ggagcagtat catgaagccc
     5161 gaaagcgaca gatctgtgtt tttgaaggca gaaagcaaaa tgtttgcttc ttttttcaaa
     5221 ggagctcgag gtggtgtctg cattccaacc actgaatccg gatcccattt gtgaataagc
     5281 cattcagact catattccct atttaacagg gtctctagtg ctgtgaaaaa aatattgctg
     5341 aacattgcat ataacttata ttgtaagaaa tactgtacat ttgaggaaga ctttattgta
     5401 cctggatagc tgtaagaaag gcatgaagga cgccaagagt tttaaggaat atagggggat
     5461 taaagtatgg agatacagaa gaaaccacta agtctgatgt ccaaatgggc acactgtcag
     5521 ttttgtttcc cttcagttgt ttgatgcatt taaaaaaaaa aaaaagaaag aaaaagaaaa
     5581 aaaggggggg gggggagaaa aaaataaatt aaaaaaaaaa aaaaaaaaag aaaagaaaga
     5641 aaaatctaag aaaaaaaaaa aaaaggttgt aggcaaatca tttgttccag gctgtgagcc
     5701 tgtgcaaaag agatttcaga tctgggcaat gggtgtgtga tctcacccac tgaagatctg
     5761 agaatgtcat ggctaggcct acatgctctg tgaatcagtc cctgtaattg ttgtttgtat
     5821 gtataattca gaagcaccaa aataagaaaa gatgtagatt tatttcatca tattatacag
     5881 actgaattgt tgtataaatt tatttactgc tagtgttagg aactgctttt tttttttttt
     5941 ttggttttaa tgtttttttt ttttttgttt tttgtttttt tttttctttc tctctggatt
     6001 tttggttgaa taaactagat tgctttcagt tgacttaagg tggatgtact ctggagggtt
     6061 tatttttcct tttattatta tttttgatgg tatttattaa atagcttcta tgggcccggc
     6121 ggtacctgtc tttttcgtca cttttcttgc agccgcggcc gccaccgcgg tggagctcca
     6181 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     6241 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     6301 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     6361 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6421 gcagcgtgac cgctacactt gccag
//
