LOCUS       sequence-412888-        4590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  this mini-donor knockin vector can be used to knock nuclear EGFP 
            into NKX2.5 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4590)
  AUTHORS   Haideri T, Lin J, Bao X, Lian XL
  TITLE     MAGIK: A rapid and efficient method to create lineage-specific 
            reporters in human pluripotent stem cells.
  JOURNAL   Stem Cell Reports. 2024 May 14;19(5):744-757. doi: 
            10.1016/j.stemcr.2024.03.005. Epub 2024 Apr 4.
  PUBMED    38579711
REFERENCE   2  (bases 1 to 4590)
  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"; doi: 
            "10.1016/j.stemcr.2024.03.005"; journalName: "Stem Cell Reports"; 
            date: "2024-05-14- 14"; volume: "19"; issue: "5"; pages: "744-757"
COMMENT     Sequence Label: mini-donor - NKX2.5-nEGFP
FEATURES             Location/Qualifiers
     source          1..4590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..145
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     137..154
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     138..154
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             742..798
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(799..801,802..804,805..1515)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(799..801,802..804,805..1515)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(844..865)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1105..1124)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1452..1473
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1516..1536
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2128..2144)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2128..2144)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2141..2163)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2152..2168
                     /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)."
     promoter        complement(join(2176..2182,2183..2200,2201..2206))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2221..2242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2359..2376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2530..3118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2610..2629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3289..4080,4081..4149))
                     /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     3912..3931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4150..4254)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4322..4340
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4378..4400)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4500..4519
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
       61 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      121 ttcccagtca cgacgttgta aaacgacggc cagtgaattg acgcgtattg ggataggaag
      181 ccgcgcgtgc tcttctcgca ggcgcaggtc tatgagctgg agcggcgctt caagcagcag
      241 cggtacctgt cggcccccga acgcgaccag ctggccagcg tgctgaaact cacgtccacg
      301 caggtcaaga tctggttcca gaaccggcgc tacaagtgca agcggcagcg gcaggaccag
      361 actctggagc tggtggggct gcccccgccg ccgccgccgc ctgcccgcag gatcgcggtg
      421 ccagtgctgg tgcgcgatgg caagccatgc ctaggggact cggcgcccta cgcgcctgcc
      481 tacggcgtgg gcctcaatcc ctacggttat aacgcctacc ccgcctatcc gggttacggc
      541 ggcgcggcct gcagccctgg ctacagctgc actgccgctt accccgccgg gccttcccca
      601 gcgcagccgg ccactgccgc cgccaacaac aacttcgtga acttcggcgt cggggacttg
      661 aatgcggttc agagccccgg gattccgcag agcaactcgg gagtgtccac gctgcatggt
      721 atccgagcct ggggaagcgg agccactaac ttctccctgt tgaaacaagc aggggatgtc
      781 gaagagaatc ccggcccaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      841 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      901 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      961 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1021 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1081 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1141 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1201 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1261 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1321 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1381 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1441 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1501 gacgagctgt acaagccaaa gaagaagcgt aaggtataac gcgaccctga ccgatcccac
     1561 ctcaacagct ccctgactct cggggggaga aggggctccc aacatgaccc tgagtcccct
     1621 ggattttgca ttcactcctg cggagaccta ggaacttttt ctgtcccacg cgcgtttgtt
     1681 cttgcgcacg ggagagtttg tggcggcgat tatgcagcgt gcaatgagtg atcctgcagc
     1741 ctggtgtctt agctgtcccc ccaggagtgc cctccgagag tccatgggca cccccggttg
     1801 gaactgggac tgagctcggg cacgcagggc ctgagatctg gccgcccatt ccgcgagcca
     1861 gggccgggcg cccgggcctt tgctatctcg ccgtcgcccg cccacgcacc cacccgtatt
     1921 tatgttttta cctattgctg taagaaatga cgatcccctt cccattaaag agagtgcgtt
     1981 gaccccgcac gtgtgcttct ttcagcttgc ggcgcttcag aagcaggaga gaggtggccg
     2041 cccgggactg gtctcagatc tcaggcacag gcattccctg agcaaattga taaatcccaa
     2101 tggcgcgccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2161 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2221 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2281 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2341 tgggcgctgt tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2401 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2461 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2521 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2581 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2641 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2701 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2761 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2821 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2881 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2941 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3001 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3061 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3121 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3181 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3241 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3301 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3361 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3421 gataccgcga gaaccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3481 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3541 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3601 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3661 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3721 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3781 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3841 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3901 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3961 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4021 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4081 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4141 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4201 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4261 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4321 aaataggcgt atcacgaggc ccttttgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     4381 ctgacacatg cagctcccgg agactgtcac agcttgtctg taagcggatg ccgggagcag
     4441 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     4501 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     4561 cgtaaggaga aaataccgca tcaggcgcca
//
