LOCUS       sequence-224403-        4918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for PCR amplification of hM3O DNA template for in vitro 
            transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4918)
  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 4918)
  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_hM3O
FEATURES             Location/Qualifiers
     source          1..4918
                     /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        1096..1114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1154..1163
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2577..2595)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2602..2619)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2602..2618)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2611..2633)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2756..3058
                     /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     2937..2956
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3407..4201
                     /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             3407..4201
                     /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(3461..3480)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4071..4090
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4401..4410
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4407..4781
                     /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 tggtaccgag ctcggatcca ctagtttgga
     1081 ccctcgtaca gaagctaata cgactcacta tagggaaata agagagaaaa gaagagtaag
     1141 aagaaatata agagccacca tggagctact gtcgccaccg ctccgcgacg tagacctgac
     1201 ggcccccgac ggctctctct gctcctttgc cacaacggac gacttctatg acgacccgtg
     1261 tttcgactcc ccggacctgc gcttcttcga agacctggac ccgcgcctga tgcacgtggg
     1321 cgcgctcctg aaacccgaag agcacatggc gggacacctg gcttcggatt tcgccttctc
     1381 gccccctcca ggtggtggag gtgatgggcc aggggggccg gagccgggct gggttgatcc
     1441 tcggacctgg ctaagcttcc aaggccctcc tggagggcca ggaatcgggc cgggggttgg
     1501 gccaggctct gaggtgtggg ggattccccc atgccccccg ccgtatgagt tctgtggggg
     1561 gatggcgtac tgtgggcccc aggttggagt ggggctagtg ccccaaggcg gcttggagac
     1621 ctctcagcct gagggcgaag caggagtcgg ggtggagagc aactccgatg gggcctcccc
     1681 ggagccctgc accgtcaccc ctggtgccgt gaagctggag aaggagaagc tggagcaaaa
     1741 cccggaggag tcccaggaca tcaaagctct gcagaaagaa ctcgagcaat ttgccaagct
     1801 cctgaagcag aagaggatca ccctgggata tacacaggcc gatgtggggc tcaccctggg
     1861 ggttctattt gggaaggtat tcagccaaac gaccatctgc cgctttgagg ctctgcagct
     1921 tagcttcaag aacatgtgta agctgcggcc cttgctgcag aagtgggtgg aggaagctga
     1981 caacaatgaa aatcttcagg agatatgcaa agcagaaacc ctcgtgcagg cccgaaagag
     2041 aaagcgaacc agtatcgaga accgagtgag aggcaacctg gagaatttgt tcctgcagtg
     2101 cccgaaaccc acactgcagc agatcagcca catcgcccag cagcttgggc tcgagaagga
     2161 tgtggtccga gtgtggttct gtaaccggcg ccagaagggc aagcgatcaa gcagcgacta
     2221 tgcacaacga gaggattttg aggctgctgg gtctcctttc tcagggggac cagtgtcctt
     2281 tcctctggcc ccagggcccc attttggtac cccaggctat gggagccctc acttcactgc
     2341 actgtactcc tcggtccctt tccctgaggg ggaagccttt ccccctgtct ctgtcaccac
     2401 tctgggctct cccatgcatt caaactgagc tgccttctgc ggggcttgcc ttctggccat
     2461 gcccttcttc tctcccttgc acctgtacct cttggtcttt gaataaagcc tggaattctg
     2521 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta
     2581 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2641 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2701 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt
     2761 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     2821 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     2881 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     2941 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
     3001 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
     3061 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
     3121 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
     3181 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     3241 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     3301 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     3361 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3421 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3481 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3541 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     3601 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3661 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3721 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3781 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3841 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3901 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     3961 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4021 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4081 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4141 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4201 aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4261 ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4321 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4381 aataatagca cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca
     4441 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
     4501 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
     4561 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
     4621 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
     4681 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
     4741 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt
     4801 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4861 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
