LOCUS       sequence-412890-        7127 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  this mini-donor knockin vector can be used to knock nuclear EGFP 
            into NKX6.1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7127)
  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 7127)
  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 - NKX6.1-nEGFP
FEATURES             Location/Qualifiers
     source          1..7127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..80
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     72..89
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     73..89
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..118
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        99..117
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1576..1632
                     /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             1633..1653
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1663..1665,1666..2379)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1663..1665,1666..2376)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1705..1726)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1966..1985)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2313..2334
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4412..4432)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4412..4430)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4451..4467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4451..4467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4464..4486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4475..4491
                     /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(4499..4505,4506..4523,4524..4529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4544..4565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4853..5441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4933..4952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5612..6403,6404..6472))
                     /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     6235..6254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6473..6577)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6603..7058)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6740..6761)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6952..6971
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
       61 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
      121 aattgggtac cgggcccccc ctcgaggtcg actcaggagt gattttgtat gtggccatta
      181 ggataccatc acagttaaaa ttattttttg cagtaggttg gtacagagcc tactgcgata
      241 atctggaggt cttcattttt caccgtaaat atgtctatat ggctagaagt gaattattat
      301 agctgtataa agaaaataaa catcactttc ttaacaattc tgattctcta gataaaatac
      361 ataatggagg acaagaataa agggaggata attcaatatc tttgggaaat atgttgcttc
      421 ttgctgatgc tttaggtaaa atatgtgtca aacatcatga ataagcttaa aaatatttgt
      481 attaaatgtc ttgagcagga acacagttaa aataacattt gaaagaatag acatgccaat
      541 tgtggaaaat aatgtaattg acaatttttc acgttttaaa aataaccaat ggtaattcta
      601 gtcttcactt tactttgggt ttatgaagaa cattaaaggc gatttgaagt ttgtatttta
      661 acagatcttt tttttttttt tttgaagtgc accatacatt gcaagtaaaa actgggagga
      721 tattttttat ctaccatgtt tgtacacatt cagatctgag ataagtgaaa caaacaaatg
      781 tttaaaagtg aaaaggagaa cagaagtaat tggggcaaat gctgtgcttt ccccccacaa
      841 tctataccga aaagcaagta cgtgtcaggt tgtttgaaat ttcttaattt aagaaagtaa
      901 ataaggaata aaatcacagc cactgtgggg caacttcatg aagggggaaa gttttcaact
      961 tcaaactgga aggcaggaaa gtgaactcac ttttagggac gctcattttt aagtttccga
     1021 gttttgtttt tacttttgag aactcgctca ctgacaacct caatactctt gtgttaccat
     1081 cgctatggca acggcagtca gtttgttggt gccagttact cagggggaac ttggggcgac
     1141 ggagactgac tttgacggcc gagaaaaggg agggctgcga gggagggcag caggccttcg
     1201 gggggccgcg ggagggccat cgagatgcgg gagtggtagc atggcctgga tctgctcgtt
     1261 taattcctcg cccttgcctc ccctctcctc ccttttctcc gccctcaggt ctggttccag
     1321 aaccgccgga ccaagtggag gaagaagcac gctgccgaga tggccacggc caagaagaag
     1381 caggactcgg agacagagcg cctcaagggg gcctcggaga acgaggaaga ggacgacgac
     1441 tacaataagc ctctggatcc caactcggac gacgagaaaa tcacgcagct gttgaagaag
     1501 cacaagtcca gcagcggcgg cggcggcggc ctcctactgc acgcgtccga gccggagagc
     1561 tcatccggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag
     1621 aaccctggac ctcccaagaa aaagcgcaag gtagcggccg cagtgagcaa gggcgaggag
     1681 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1741 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1801 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1861 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1921 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1981 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2041 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2101 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2161 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2221 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2281 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2341 gccgggatca ctctcggcat ggacgagctg tacaagtgat gaacgccgcc gcccgccgca
     2401 ccttcccggc tccggcctcc acctctgggg ccgcgagggg cgcggggacc gcgtgcacgc
     2461 cgcgcctgcc cgctcctcgg ggcccggagg acccggccct gcctgcgggg accctttgct
     2521 atttttgaga tgtacatatc tattttttta accttgaagt tgtgggaggg gatgtagggg
     2581 agagagactc cggggttgat aaggcaagaa agaccaccaa atgcactgcg taaatgtcgc
     2641 tactgaaacc cgcagccggg ccacggtccc agccgcgtcc ccataggcgg gcagcggcct
     2701 cagcgcccgg gtccccctcg cccccagatt ttgcccccgc gggcggcggg gtagggggcg
     2761 ccgagaggtg ctgtggaagg gttcacgctc accgcatggg ggacctcgga gcgaagcggg
     2821 cgggcgctgc cacagatggg aagagggacg tttccccccc cacacactgc tgtgcaacta
     2881 aagtaacctg ttgaaggctc tttgtaaata aatcgtgagt tacactgact agaagttact
     2941 ttattgtgaa tatttaaaat gtaaaatgct tttttgaatt tcgtataaaa agatggactt
     3001 tcttccttct cctcctcccc tttgaagagc gactcaagcc acaccttccc ttttattttg
     3061 aacttaattt gagaagcgta gaatagaatg tccccacctt ctccgtcccc tcctctcgtt
     3121 cctctttccc gtttggattt gcagaatctc acctttgccc ttcatctctt ctcgccctgc
     3181 cctgccctgc ccaccccctg gatctacact gcgctcccgc cccccggact tgaagtggaa
     3241 gacgtgggaa ccagggaggg cctctcacct gcctccctgt tcctctctga aattgcacag
     3301 tcgtttgttg ctatttatta gtgatttaaa gaaaatattg gggagggagg ggctacaata
     3361 gcttgtattt aactcctttc tgataaaaat gcgtgttaaa gtaggtggtg ggaattgtta
     3421 caaccgctct gggtctgaaa cataaaaatt catttagtta ctgtaattga atttgaagtg
     3481 ttttgtatca taagaatact atagaatatg taaattgttt tctttttaca gctaataatg
     3541 gtgatatatt agcatcttta acctttatta atttatataa ggaattcggt ttgtaaagag
     3601 aagaaaaccc cctttgcaag accagttaaa tgtaatgcac tttctgtttc aaaggataaa
     3661 tcaaattaaa aaaaacagtt tgaagacgtc tgttgccttg aatgaaaggg tacagatcat
     3721 ctgatctcat cagagatctc aaataataaa agtcaagacc acaaaaaaaa tggtatatcg
     3781 gtttcttggg tgaaatagct gttacttaat ttttttttct ttggatgagt gatgtcacat
     3841 ttctcattct cctggatagg aaaataaaat aaatgcagcc ccactcatgg tgattcttgg
     3901 agagactagt ctagctagct agctgcttct gatgtctgtg ttgtctaatt tcttcctgat
     3961 cgctttttcc ttttccagga agtccctggg gttggggggc agttgggaag agctcatttc
     4021 catatatcca agatagctcc tcagtcagcc cctattcgat tagcgtagag gaggcagcgg
     4081 agctcgctgg ttgagtagca ctgctgtttg atgctaatat ttctcccccg tagctgtaag
     4141 actgcagaaa ttgtggaaag acagaagcct tgattttact aaacattact gttgaaagat
     4201 gaatttaaag aaagcttcct ttaactggtg taaaaatatt ttagggttaa gaaagcaatg
     4261 tcatagcttc tccagctgcc gaatctgaag aaaaaaaaaa ttgggaaggc cagggctgta
     4321 tcactgtttt gggatctttt tgtttttaga cactcggttt caggctcagt ctagagcggc
     4381 cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     4441 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4501 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4561 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4621 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4681 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4741 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4801 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4861 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4921 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4981 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5041 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5101 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5161 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5221 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5281 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5341 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5401 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5461 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5521 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5581 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5641 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5701 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5761 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5821 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5881 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5941 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6001 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6061 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6121 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6181 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6241 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6301 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6361 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6421 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6481 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6541 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6601 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     6661 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     6721 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     6781 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     6841 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     6901 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     6961 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     7021 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     7081 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgcc
//
