LOCUS       sequence-217796-        6450 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  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 6450)
  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..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    45..66
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(81..86,87..104,105..111)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    119..135
                     /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     124..146
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     143..159
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     143..159
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     178..198
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        180..198
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     227..243
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     228..244
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1483..1536
                     /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             1543..1920
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1933..1942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1939..1941,1942..1944,1945..2658)
                     /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(1939..1941,1942..1944,1945..2655)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1984..2005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2245..2264)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2592..2613
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2660..2676)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2660..2676)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3815..3834)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3816..3834)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3844..3861)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3844..3860)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3853..3875)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4001..4456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4138..4159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4350..4369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4483..4587
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4588..4656,4657..5448)
                     /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(4806..4825)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5619..6207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6108..6127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6361..6378
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
       61 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      121 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      181 attaaccctc actaaaggga acaaaagctg ggtaccgggc cccccctcga ggtcgacggt
      241 atcgataagc ttccagcatg tcctacaaat ctccagagta gggctttgca tctttgcctg
      301 atgcattcat gttttttttt ttttcttgtg accgtgagta tccttacaag ccatggaagg
      361 actttgggtc ctgttggctg ttgttaaatc cttgttgatt ggatggcttg agttgcttgt
      421 gcagaagcag gaaggggaaa gcaacagcct tgaagcactg cagctgtgtt gggtgatgtg
      481 ggcacagggt tatcctttgg aagagcatac tgctttagca caggctccgg agctgtggat
      541 gtcgctccct ggcttccacc cctaggaccc tattccctcc aaaaatctca gaacacacac
      601 atttccaggg gaagtttcca gaacatcacc ctgtgaaaag gccaggtaga agagtgacag
      661 ccagaccttg caggaaggca cagagaccca gttttctaga aaggtggatt tcaacacagg
      721 aggcttcact ctgtctggct ctgtgtggca agtcccagag aagactcatc atatacgtaa
      781 acaatggtgt ctggcgaccc tgattcccca tcttctcatg aaatatctta gggaaatctt
      841 aaggaagtaa cacaggttga ggaagtaaca caggtttttg tattttagtc atcagatata
      901 tactcgatac taggtgggcc ccgaacttcc cctgttgatg agtttagctg aattgtgctc
      961 ggggcctgtg ggctttgaaa gcaatggccc ctgaatcaga agccctgtgt tccaaggcct
     1021 gcagtacctg ggtctctgga aagaaggaag gagtaggtag gaagaagagg caatcaacac
     1081 aggaacacat ccctggcctg gggcctgtgc tactgtgcaa aagcttttat catttcaaag
     1141 gcagcaaaaa agtaaaaaaa aaaaaaattg atctttgttt agattaacag acccctgact
     1201 atgaagaaag aaggcatcca gacccgaaac cggaagatgt ctagcaaatc gaaaaagtgc
     1261 aaaaaggtgc atgacgcgct ggaggacttc cccaagagca gctccttcaa cccggccgct
     1321 ctctccagac acatgtcatc cctgagccac atctctccct tcagccactc cagccacatg
     1381 ctgaccacac cgacgcccat gcatccgccc tccggcctct ccttcggacc tcaccaccct
     1441 tccagcatgg tcaccgccat gggtaagctt gatatcgaat tcgagggcag aggaagtctt
     1501 ctaacatgcg gtgacgtgga ggagaatccc ggccctaccg gtatgccaga gccagcgaag
     1561 tctgctcccg ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaaggc
     1621 ggcaagaagc gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg
     1681 aagcaggtcc accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt
     1741 gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag
     1801 cgctcgacca tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag
     1861 ttggccaagc acgccgtgtc cgagggtact aaggccatca ccaagtacac cagcgctaag
     1921 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1981 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2041 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2101 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2161 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2221 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2281 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2341 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2401 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2461 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2521 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2581 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2641 gacgagctgt acaagtaagg atccactagt tctagagcgg ccgcagaggc agagccctgc
     2701 tccacatgcg tgaggagtct ccaagtgtgc gaagagttcc tccgacccct tctacttgcg
     2761 tttttcgcag gagcagtatc atgaagcccg aaagcgacag atctgtgttt ttgaaggcag
     2821 aaagcaaaat gtttgcttct tttttcaaag gagctcgagg tggtgtctgc attccaacca
     2881 ctgaatccgg atcccatttg tgaataagcc attcagactc atattcccta tttaacaggg
     2941 tctctagtgc tgtgaaaaaa atattgctga acattgcata taacttatat tgtaagaaat
     3001 actgtacatt tgaggaagac tttattgtac ctggatagct gtaagaaagg catgaaggac
     3061 gccaagagtt ttaaggaata tagggggatt aaagtatgga gatacagaag aaaccactaa
     3121 gtctgatgtc caaatgggca cactgtcagt tttgtttccc ttcagttgtt tgatgcattt
     3181 aaaaaaaaaa aaaaraaara aaaaraaaaa aagggggggg ggggaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaga aaagaaagaa aaatctaaga aaaaaaaaaa aaaggttgta
     3301 ggcaaatcat ttgttccagg ctgtgagcct gtgcaaaaga gatttcagat ctgggcaatg
     3361 ggtgtgtgat ctcacccact gaagatctga gaatgtcatg gctaggccta catgctctgt
     3421 gaatcagtcc ctgtaattgt tgtttgtatg tataattcag aagcaccaaa ataagaaaag
     3481 atgtagattt atttcatcat attatacaga ctgaattgtt gtataaattt atttactgct
     3541 agtgttagga actgcttttt tttttttttt tggttttaat gttttttttt tttttgtttt
     3601 ttkttttttt ttttytttyt ctctggattt ttggttgaat aaactagatt gctttcagtt
     3661 gacttaaggt ggatgtactc tggagggttt atttttcctt ttattattat ttttgatggt
     3721 atttattaaa tagcttctat gggcccggcg gtacctgtct ttttcgtcac ttttcttgca
     3781 gccgcggccg ccaccgcggt ggagctccaa ttcgccctat agtgagtcgt attacgcgcg
     3841 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3901 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3961 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt
     4021 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4081 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4141 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4201 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4261 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4321 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4381 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4441 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4501 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4561 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4621 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4681 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4741 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4801 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4861 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4921 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4981 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5041 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5101 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5161 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5221 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5281 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5341 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5401 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5461 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5521 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5581 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5641 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5701 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5761 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5821 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5881 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5941 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6001 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6061 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6121 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6181 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6241 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6301 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6361 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     6421 cgttggccga ttcattaatg cagctggcac
//
