LOCUS       sequence-8512-ex        2706 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2706)
  AUTHORS   Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B
  TITLE     Facile methods for generating human somatic cell gene knockouts 
            using recombinant adeno-associated viruses.
  JOURNAL   Nucleic Acids Res. 2004  . 32(1):e3.
  PUBMED    14704360
REFERENCE   2  (bases 1 to 2706)
  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: "Nucleic 
            Acids Res. 2004  . 32(1):e3."
COMMENT     Sequence Label: pNeDaKO-Hyg
FEATURES             Location/Qualifiers
     source          1..2706
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    complement(37..70)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        82..582
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(117..140)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     515..534
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             599..1618
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             599..1618
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(1926..1943)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1932..2156
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2197..2244
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        2236..2254
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2258..2267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2264..2638
                     /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"
     protein_bind    complement(2639..2672)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(2687..2705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctaaagggaa caaaagctgg agctccaccg cggtggataa cttcgtatag catacattat
       61 acgaagttat cgcgccctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
      121 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
      181 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
      241 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
      301 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
      361 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttggctc cttcgctttc
      421 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc
      481 ggggcgggcg cccgaaggtc ctccggaagc ccggcattct gcacgcttca aaagcgcacg
      541 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgaatggata gatccggaaa
      601 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
      661 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
      721 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
      781 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
      841 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
      901 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
      961 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     1021 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     1081 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     1141 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     1201 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     1261 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     1321 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     1381 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     1441 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     1501 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     1561 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag
     1621 tagatgccga ccgaacaaga gctgatttcg agaacgcctc agccagcaac tcgcgcgagc
     1681 ctagcaaggc aaatgcgaga gaacggcctt acgcttggtg gcacagttct cgtccacagt
     1741 tcgctaagct cgctcggctg ggtcgcggga gggccggtcg cagtgattca ggcccttctg
     1801 gattgtgttg gtccccaggg cacgattgtc atgcccacgc actcgggtga tctgactgat
     1861 cccgcagatt ggagatcgcc gcccgtgcct gccgattggg tgcagatcta gagctcgctg
     1921 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1981 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2041 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2101 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2161 tgaggcggaa agaaccagct gggggctgca gcacgtgttg acaattaatc atcggcatag
     2221 tatatcggca tagtataata cgactcacta taggagggcc accatggcca agttgaccag
     2281 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     2341 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     2401 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     2461 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     2521 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     2581 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaat
     2641 aacttcgtat agcatacatt atacgaagtt atggtaccca attcgcccta tagtgagtcg
     2701 tattac
//
