LOCUS       sequence-246236-        1825 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  destination vector with N-terminal V5 tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1825)
  AUTHORS   Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D,
            Eggan K, Barrett LE
  TITLE     A multiplexed gRNA piggyBac transposon system facilitates efficient 
            induction of CRISPRi and CRISPRa in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2020 Jan 20;10(1):635. doi: 10.1038/s41598-020-57500-1.
  PUBMED    31959800
REFERENCE   2  (bases 1 to 1825)
  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: "Sci Rep."; 
            date: "2020-01-20"; volume: "10(1):635. doi"; pages: " 
            10.1038/s41598-020-57500-1"
COMMENT     Sequence Label: S1_AscI_V5-gateway_Bsu36I
FEATURES             Location/Qualifiers
     source          1..1825
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      55..64
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             64..105
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    106..230
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(255..260,261..278,279..285)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             339..998
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(405..424)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1340..1645
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1524..1543
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1686..1810)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ggcgcgccta tcgaaattca ctatagggag acccaagctg gctagttaag cttcgccacc
       61 atgggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgacaag tttgtacaaa
      121 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
      181 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat
      241 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt
      301 aggatccggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat
      361 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag
      421 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg
      481 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga
      541 atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg
      601 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg
      661 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg
      721 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca
      781 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg
      841 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg
      901 cgattcaggt tcatcatgcc gtctgtgatg gcttccatgt cggcagaatg cttaatgaat
      961 tacaacagta ctgcgatgag tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa
     1021 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     1081 atatgtatac ccgaagtatg tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac
     1141 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     1201 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa
     1261 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     1321 gagaacaggg actggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     1381 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     1441 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     1501 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     1561 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     1621 taacctgatg ttctggggaa tataaatgtc aggctccgtt atacacagcc agtctgcagg
     1681 tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     1741 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt
     1801 acaaagtggt agtaatgacc ttagg
//
