LOCUS       sequence-115398-        4969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PDX1-eGFP mini-vector donor for integration of the eGFP reporter 
            into the PDX1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4969)
  AUTHORS   Zhu Z, Verma N, Gonzalez F, Shi ZD, Huangfu D
  TITLE     A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human 
            Embryonic Stem Cells.
  JOURNAL   Stem Cell Reports. 2015 May 28. pii: S2213-6711(15)00128-9. doi: 
            10.1016/j.stemcr.2015.04.016.
  PUBMED    26028531
REFERENCE   2  (bases 1 to 4969)
  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: "Stem Cell 
            Reports. 2015 May 28. pii: S2213-6711(15)00128-9. doi: 
            10.1016/j.stemcr.2015.04.016."
COMMENT     Sequence Label: PDX1-2A-eGFP
FEATURES             Location/Qualifiers
     source          1..4969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1411..1467
                     /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(1468..1470,1471..1473,1474..2187)
                     /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(1468..1470,1471..1473,1474..2184)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1513..1534)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1774..1793)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2121..2142
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2781..2801)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2781..2799)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2820..2836)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2820..2836)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2833..2855)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2844..2860
                     /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(2868..2874,2875..2892,2893..2898))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2913..2934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3051..3068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3222..3810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3302..3321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3981..4772,4773..4841))
                     /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     4604..4623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4842..4946)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgaca ggaccactca ttggcagagc caagcttagg ctcacggcga
      721 gagctgactc gagtttggtc tccaataaaa aggctatctt tattaggaag ggcttgagtt
      781 actagggaag agcttcgcgc gcctacacta ggcgctgaaa tgggatgctg gggcttggtg
      841 gctccggcgg gagcagctgg tagggctagg gctccctggc cccccttgaa gcggttgggc
      901 tgcgtgggtg ggggctgtgc ggggctccgg gggccacact cacgccctgt gtcgcccgca
      961 ggcggcgcct acgctgcgga gccggaggag aacaagcgga cgcgcacggc ctacacgcgc
     1021 gcacagctgc tagagctgga gaaggagttc ctattcaaca agtacatctc acggccgcgc
     1081 cgggtggagc tggctgtcat gttgaacttg accgagagac acatcaagat ctggttccaa
     1141 aaccgccgca tgaagtggaa aaaggaggag gacaagaagc gcggcggcgg gacagctgtc
     1201 gggggtggcg gggtcgcgga gcctgagcag gactgcgccg tgacctccgg cgaggagctt
     1261 ctggcgctgc cgccgccgcc gccccccgga ggtgctgtgc cgcccgctgc ccccgttgcc
     1321 gcccgagagg gccgcctgcc gcctggcctt agcgcgtcgc cacagccctc cagcgtcgcg
     1381 cctcggcggc cgcaggaacc acgagctagc gccactaact tctccctgtt gaaacaagca
     1441 ggggatgtcg aagagaatcc cgggccaatg gtgagcaagg gcgaggagct gttcaccggg
     1501 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1561 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1621 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1681 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1741 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1801 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1861 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1921 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1981 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2041 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2101 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2161 ctcggcatgg acgagctgta caagtaaggc gcgccgaggc aggagctgct cctggctgag
     2221 gggcttcaac cactcgccga ggaggagcag agggcctagg aggaccccgg gcgtggacca
     2281 cccgccctgg cagttgaatg gggcggcaat tgcggggccc accttagacc gaaggggaaa
     2341 acccgctctc tcaggcgcat gtgccagttg gggccccgcg ggtagatgcc ggcaggcctt
     2401 ccggaagaaa aagagccatt ggtttttgta gtattggggc cctcttttag tgatactgga
     2461 ttggcgttgt ttgtggctgt tgcgcacatc cctgccctcc tacagcactc caccttggga
     2521 cctgtttaga gaagccggct cttcaaagac aatggaaact gtaccataca cattggaagg
     2581 ctccctaaca cacacagcgg ggaagctggg ccgagtacct taatctgcca taaagccatt
     2641 cttactcggg cgaccccttt aagtttagaa ataattgaaa ggaaatgttt gagttttcaa
     2701 agatcccgtg aaattgatgc cagtggaata cagtgagttc tagagcggcc gccaccgcgg
     2761 tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     2821 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2881 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2941 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3001 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3061 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3121 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3661 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3901 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3961 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4021 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4081 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4141 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4201 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4261 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4321 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4381 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4441 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4501 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4561 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4621 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4681 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4741 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4801 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4861 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
