LOCUS       sequence-224405-        4549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for PCR amplification of hNANOG DNA template for in vitro 
            transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4549)
  AUTHORS   McGrath PS, Diette N, Kogut I, Bilousova G
  TITLE     RNA-based Reprogramming of Human Primary Fibroblasts into Induced 
            Pluripotent Stem Cells.
  JOURNAL   J Vis Exp. 2018 Nov 26;(141). doi: 10.3791/58687.
  PUBMED    30531717
REFERENCE   2  (bases 1 to 4549)
  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: "J Vis 
            Exp."; date: "2018-11-26"; volume: "(141). doi"; pages: " 
            10.3791/58687"
COMMENT     Sequence Label: pCR-Blunt_hNANOG
FEATURES             Location/Qualifiers
     source          1..4549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1078..1096
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2208..2226)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2233..2250)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2233..2249)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2242..2264)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2387..2689
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2568..2587
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3038..3832
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3038..3832
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3092..3111)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3702..3721
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4032..4041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4038..4412
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgacg
     1021 cgttagaata ctcaagctat gcatcaagct tggtaccttg gaccctcgta cagaagctaa
     1081 tacgactcac tatagggaaa taagagagaa aagaagagta agaagaaata taagagccac
     1141 catgagtgtg gatccagctt gtccccaaag cttgccttgc tttgaagcat ccgactgtaa
     1201 agaatcttca cctatgcctg tgatttgtgg gcctgaagaa aactatccat ccttgcaaat
     1261 gtcttctgct gagatgcctc acacggagac tgtctctcct cttccttcct ccatggatct
     1321 gcttattcag gacagccctg attcttccac cagtcccaaa ggcaaacaac ccacttctgc
     1381 agagaagagt gtcgcaaaaa aggaagacaa ggtcccggtc aagaaacaga agaccagaac
     1441 tgtgttctct tccacccagc tgtgtgtact caatgataga tttcagagac agaaatacct
     1501 cagcctccag cagatgcaag aactctccaa catcctgaac ctcagctaca aacaggtgaa
     1561 gacctggttc cagaaccaga gaatgaaatc taagaggtgg cagaaaaaca actggccgaa
     1621 gaatagcaat ggtgtgacgc agaaggcctc agcacctacc taccccagcc tttactcttc
     1681 ctaccaccag ggatgcctgg tgaacccgac tgggaacctt ccaatgtgga gcaaccagac
     1741 ctggaacaat tcaacctgga gcaaccagac ccagaacatc cagtcctgga gcaaccactc
     1801 ctggaacact cagacctggt gcacccaatc ctggaacaat caggcctgga acagtccctt
     1861 ctataactgt ggagaggaat ctctgcagtc ctgcatgcag ttccagccaa attctcctgc
     1921 cagtgacttg gaggctgcct tggaagctgc tggggaaggc cttaatgtaa tacagcagac
     1981 cactaggtat tttagtactc cacaaaccat ggatttattc ctaaactact ccatgaacat
     2041 gcaacctgaa gacgtgtgag ctgccttctg cggggcttgc cttctggcca tgcccttctt
     2101 ctctcccttg cacctgtacc tcttggtctt tgaataaagc ctggaattct gcagatatcc
     2161 atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct atagtgagtc
     2221 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2281 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2341 ccgcaccgat cgcccttccc aacagttgcg cagcctatac gtacggcagt ttaaggttta
     2401 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
     2461 cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
     2521 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac
     2581 cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg
     2641 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcat
     2701 gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca gtccgcagaa
     2761 acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag
     2821 cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt
     2881 tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa
     2941 gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc
     3001 aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3061 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3121 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3181 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     3241 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3301 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3361 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3421 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3481 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3541 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     3601 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3661 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3721 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3781 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattattaa
     3841 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3901 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3961 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc
     4021 acgtgaggag ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     4081 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     4141 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     4201 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     4261 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     4321 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     4381 gcgtgcactt cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta
     4441 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
