LOCUS       sequence-217821-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in eGFP fusing to the c-terminal of mouse 
            Gata4 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  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 6714)
  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: Gata4-GFP donor
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2048..2050,2051..2053,2054..2767)
                     /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(2048..2050,2051..2053,2054..2764)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2093..2114)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2354..2373)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2701..2722
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3804..3820)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3804..3820)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3852..3871)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3853..3871)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3881..3898)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3881..3897)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3890..3912)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4038..4493)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4175..4196)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4387..4406
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4520..4624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4625..4693,4694..5485)
                     /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(4843..4862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5656..6244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6145..6164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6398..6415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6532..6553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6568..6573,6574..6591,6592..6598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6606..6622
                     /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     6611..6633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6630..6646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6630..6646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6665..6685
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6667..6685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     join(6714,1..16)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 cgaggtcgac ggtatcgata agcttgatat cgaattccag actgtgacta cagcagagcc
       61 cctgggtaac ctccctcatc ctgtgtggca ggcacaggaa gaacaggcac tgggttcagt
      121 ggagtagccc tcaaccccta agtggaaatc cagaaaagta aattccatag cagaatctgt
      181 tctggagagc acagggcatt tttaatctcg actcatccca agcagtgtgg ctatccacac
      241 ttcactacag agaaattaac acagttaatt atgagttggc gtcccagaac cctctggcag
      301 tatcctgcaa tatatagtat ttgcatttaa gaagcagtat caagagtgta gaatcccaaa
      361 atacaacgtg gtctccagga tcgcctctcc tggatctagg gtcgtgaggg aatcagagac
      421 ttttgaaaag gttcccttga ggctggtgat gcagctcggt gggtagggtg cttacttagc
      481 atgcacaaag tcctggcttc agcccccagc attccataaa ccgggcatgg cggcagaggc
      541 ttgtaaaccc agcccttggg aggcggaggc aggaatgtca gttcagtggc atccttacct
      601 acacagtgag tttgtgacca gcctgggctg cagcggtttt tacaagaagg taaaatggtc
      661 tccttgggat tttgatgctt ctgccctatt ttgagaactg ctccaccccc caacccagca
      721 agtataaaaa aggcctctgt ccctgtagct ggactgggtc tcctgactct gcctcctcct
      781 ttgcaggtcc tgctggtgag accctccctc cctccagtgg tgcctccagc ggtaactcca
      841 gcaatgccac tagcagcagc agcagcagtg aagagatgcg ccccatcaag acagagcccg
      901 ggctgtcatc tcactatggg cacagcagct ccatgtccca ggtgggcagg gggacggagt
      961 tgatcccagt agccagtctt gccttgggaa atgagctgta gactggccct aagctattgg
     1021 cctccaagca gcccagcctc tgagcccttg gtacctcttg tccagctcca ctgatttaga
     1081 cacaaagaat atcacaggct taaggtttag cgccttcctg tgctgggtgg ggcctttggg
     1141 aacaaggaga aggactcagg gactgcaccc ggggtgcatg tggcagatag tacaactgct
     1201 ccctgagtca gtcttgagtg ttggggttgg aagcagattt ggactctaga gcttgcagag
     1261 ctttgagcct agccttgaag gattttcaga ggaggaggca ggaagacggt gtgagcatgt
     1321 catagacttg ggaaccacag agggtgggaa gctgttttca tgagctctag gttcccgaga
     1381 gcctccgagt ggccctacaa gggaccgtgc cctcttgcag gtctgatggc acctctgttc
     1441 ctctgccttt taggggatga ggaagtcaca ggttcccttt cccagcactg ccacccaatg
     1501 gttgagaatt ctaccatcag agtaaactag agagaacctg gctccacaga agtgagagtg
     1561 ctagaaccct ttcaggtgga gcgggtgacc tcagttggac ccagaagacc tctatctgac
     1621 cagggagtgg tgataagagt gtctcctgtg gctgccagag ctttctgaga aagcacagca
     1681 gcacaaaact cctgggttgt acctctggtt acagtgctca gaagccaacg tggaagcaca
     1741 cagaccctct gcggctggac cctagggcct agggaagcag ctagtcttac tccctcattc
     1801 cctccccaac cctggtcctc ggatctccag gagcatccta tggctcactg tgctctctgc
     1861 cttccagaca ttcagtactg tgtccggcca cgggccctcc atccatccag tgctgtctgc
     1921 tctgaagctg tccccacaag gctatgcatc tcctgtcact cagacatcgc aggccagctc
     1981 caagcaggac tcttggaaca gcctggtcct ggctgacagt catggggaca taatcaccgc
     2041 ggaattcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2101 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2161 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2221 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2281 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2341 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2401 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2461 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2521 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2581 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2641 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2701 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2761 caagtgaccc gggtcagcgc ccccccttcc ctcttcaaat tcctgctcgg acttgggacg
     2821 tgggggccag caaagtaaaa ggctggggca cccttggcca gcccctttgt ctgggaacaa
     2881 ctcctgaaga acaactgggt agaacttgaa gttgttgaca atcacttagg gatatgggtg
     2941 ttccgggttg ttcaaacacc tttccaggtg gagcactgga aaagcctgcg ttcttacaga
     3001 gaagcccacc ttggctgcaa gcacagcaca gtgaggcaag agacttcttc cttccttatt
     3061 ctccacctgc ctgtccagga cagacacata atctccttca ccccagctcc ccacccagtt
     3121 gtggtggtgg gtttttcttt gtgatcctag agtggctgta ggggcggagg cttcaagaca
     3181 ccatctacag tctgagcagg gtgtctactt gttgtagact agacatagaa gccctgccct
     3241 tgtccaacac tccccttgct tgaggcatgg cacatctctg catgtcccat accagatctg
     3301 actccaaagt gctgggttca atgcagatgt tactgaatgc ttcctgggga gattaggtga
     3361 ggggaaggca catcacccat cacacagaat agcttcatca aatcgcagcc tggccatggt
     3421 gccttccctt cctctcccag gaacatcaaa ccccttgctc tccagcctga acatctaccc
     3481 tctgcaaaag tagagcccag ttgtgcagct aatgccacta ggtgctatat cccagcatcc
     3541 ttttcacccc ttcacacaca ggggttccaa ggaggaacaa aacctgctac caaagcagcc
     3601 ttggtgacta tggctcatct gcacctcagg gggtggggga gggccctctg gaggttgtgt
     3661 ctacagcaca atactgttcc caggactcta gcttgcttgc cccgagcctg ccaagccaag
     3721 ccctcttaag tcagacagtt acctggctct gggactttct ccagcacaga tcctttgtct
     3781 agaaaataca gactgtcccg ggggatccac tagttctaga gcggccgcca ccgcggtgga
     3841 gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3901 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3961 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4021 gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4081 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4141 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4201 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4261 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4321 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4381 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4441 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     4501 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4561 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4621 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4681 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4741 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4801 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4861 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4921 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4981 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5041 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5101 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5161 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5221 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5281 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5341 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5401 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5461 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5521 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5581 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5761 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6301 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6361 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6421 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     6481 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     6541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     6661 gcgcgcaatt aaccctcact aaagggaaca aaagctgggt accgggcccc ccct
//
