LOCUS       sequence-213386-        7026 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Donor plasmid for knock-in eGFP fusing to the c-terminal of mouse 
            Gata4 coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7026)
  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 7026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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..7026
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(5..21)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(54..72)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(82..98)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      240..695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(327..346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     537..558
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        721..825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(826..894,895..1686)
                     /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(1044..1063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1857..2445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2346..2365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2599..2616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2733..2754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2769..2774,2775..2792,2793..2799)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2807..2823
                     /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     2812..2834
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2831..2847
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2831..2847
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2866..2886
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2868..2886
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2915..2931
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2916..2932
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(4963..4965,4966..4968,4969..5682)
                     /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(4963..4965,4966..4968,4969..5679)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5008..5029)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5269..5288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5616..5637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6719..6735)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(6768..6786)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6796..6812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggggatcca ctagttctag agcggccgcc accgcggtgg agctccaatt cgccctatag
       61 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
      121 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
      181 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
      241 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      301 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
      361 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
      421 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
      481 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
      541 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
      601 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
      661 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
      721 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      781 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      841 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      901 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      961 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1021 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1081 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1141 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1201 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1261 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1321 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1381 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1441 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1501 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     1561 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1621 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1681 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1741 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1801 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1861 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1921 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1981 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2041 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2101 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2161 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2221 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2281 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2341 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2401 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2461 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2521 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2581 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     2641 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     2701 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     2761 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     2821 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     2881 taaagggaac aaaagctggg taccgggccc cccctcgagg tcgacggtat cgataagctt
     2941 gatatcgaat tccagactgt gactacagca gagcccctgg gtaacctccc tcatcctgtg
     3001 tggcaggcac aggaagaaca ggcactgggt tcagtggagt agccctcaac ccctaagtgg
     3061 aaatccagaa aagtaaattc catagcagaa tctgttctgg agagcacagg gcatttttaa
     3121 tctcgactca tcccaagcag tgtggctatc cacacttcac tacagagaaa ttaacacagt
     3181 taattatgag ttggcgtccc agaaccctct ggcagtatcc tgcaatatat agtatttgca
     3241 tttaagaagc agtatcaaga gtgtagaatc ccaaaataca acgtggtctc caggatcgcc
     3301 tctcctggat ctagggtcgt gagggaatca gagacttttg aaaaggttcc cttgaggctg
     3361 gtgatgcagc tcggtgggta gggtgcttac ttagcatgca caaagtcctg gcttcagccc
     3421 ccagcattcc ataaaccggg catggcggca gaggcttgta aacccagccc ttgggaggcg
     3481 gaggcaggaa tgtcagttca gtggcatcct tacctacaca gtgagtttgt gaccagcctg
     3541 ggctgcagcg gtttttacaa gaaggtaaaa tggtctcctt gggattttga tgcttctgcc
     3601 ctattttgag aactgctcca ccccccaacc cagcaagtat aaaaaaggcc tctgtccctg
     3661 tagctggact gggtctcctg actctgcctc ctcctttgca ggtcctgctg gtgagaccct
     3721 ccctccctcc agtggtgcct ccagcggtaa ctccagcaat gccactagca gcagcagcag
     3781 cagtgaagag atgcgcccca tcaagacaga gcccgggctg tcatctcact atgggcacag
     3841 cagctccatg tcccaggtgg gcagggggac ggagttgatc ccagtagcca gtcttgcctt
     3901 gggaaatgag ctgtagactg gccctaagct attggcctcc aagcagccca gcctctgagc
     3961 ccttggtacc tcttgtccag ctccactgat ttagacacaa agaatatcac aggcttaagg
     4021 tttagcgcct tcctgtgctg ggtggggcct ttgggaacaa ggagaaggac tcagggactg
     4081 cacccggggt gcatgtggca gatagtacaa ctgctccctg agtcagtctt gagtgttggg
     4141 gttggaagca gatttggact ctagagcttg cagagctttg agcctagcct tgaaggattt
     4201 tcagaggagg aggcaggaag acggtgtgag catgtcatag acttgggaac cacagagggt
     4261 gggaagctgt tttcatgagc tctaggttcc cgagagcctc cgagtggccc tacaagggac
     4321 cgtgccctct tgcaggtctg atggcacctc tgttcctctg ccttttaggg gatgaggaag
     4381 tcacaggttc cctttcccag cactgccacc caatggttga gaattctacc atcagagtaa
     4441 actagagaga acctggctcc acagaagtga gagtgctaga accctttcag gtggagcggg
     4501 tgacctcagt tggacccaga agacctctat ctgaccaggg agtggtgata agagtgtctc
     4561 ctgtggctgc cagagctttc tgagaaagca cagcagcaca aaactcctgg gttgtacctc
     4621 tggttacagt gctcagaagc caacgtggaa gcacacagac cctctgcggc tggaccctag
     4681 ggcctaggga agcagctagt cttactccct cattccctcc ccaaccctgg tcctcggatc
     4741 tccaggagca tcctatggct cactgtgctc tctgccttcc agacattcag tactgtgtcc
     4801 ggccacgggc cctccatcca tccagtgctg tctgctctga agctgtcccc acaaggctat
     4861 gcatctcctg tcactcagac atcgcaggcc agctccaagc aggactcttg gaacagcctg
     4921 gtcctggctg acagtcatgg ggacataatc accgcggaat tcatggtgag caagggcgag
     4981 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     5041 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     5101 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     5161 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     5221 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     5281 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     5341 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     5401 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     5461 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     5521 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     5581 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     5641 gccgccggga tcactctcgg catggacgag ctgtacaagt gacccgggtc agcgcccccc
     5701 cttccctctt caaattcctg ctcggacttg ggacgtgggg gccagcaaag taaaaggctg
     5761 gggcaccctt ggccagcccc tttgtctggg aacaactcct gaagaacaac tgggtagaac
     5821 ttgaagttgt tgacaatcac ttagggatat gggtgttccg ggttgttcaa acacctttcc
     5881 aggtggagca ctggaaaagc ctgcgttctt acagagaagc ccaccttggc tgcaagcaca
     5941 gcacagtgag gcaagagact tcttccttcc ttattctcca cctgcctgtc caggacagac
     6001 acataatctc cttcacccca gctccccacc cagttgtggt ggtgggtttt tctttgtgat
     6061 cctagagtgg ctgtaggggc ggaggcttca agacaccatc tacagtctga gcagggtgtc
     6121 tacttgttgt agactagaca tagaagccct gcccttgtcc aacactcccc ttgcttgagg
     6181 catggcacat ctctgcatgt cccataccag atctgactcc aaagtgctgg gttcaatgca
     6241 gatgttactg aatgcttcct ggggagatta ggtgagggga aggcacatca cccatcacac
     6301 agaatagctt catcaaatcg cagcctggcc atggtgcctt cccttcctct cccaggaaca
     6361 tcaaacccct tgctctccag cctgaacatc taccctctgc aaaagtagag cccagttgtg
     6421 cagctaatgc cactaggtgc tatatcccag catccttttc accccttcac acacaggggt
     6481 tccaaggagg aacaaaacct gctaccaaag cagccttggt gactatggct catctgcacc
     6541 tcagggggtg ggggagggcc ctctggaggt tgtgtctaca gcacaatact gttcccagga
     6601 ctctagcttg cttgccccga gcctgccaag ccaagccctc ttaagtcaga cagttacctg
     6661 gctctgggac tttctccagc acagatcctt tgtctagaaa atacagactg tcccggggga
     6721 tccactagtt ctagagcggc cgccaccgcg gtggagctcc aattcgccct atagtgagtc
     6781 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     6841 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     6901 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     6961 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     7021 tgccag
//
