LOCUS       sequence-254527-        4451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  destination vector with N-terminal V5 tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4451)
  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 4451)
  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..4451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..55)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     155..174
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     368..390
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     382..399
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     383..399
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      474..483
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             483..524
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    525..649
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(674..679,680..697,698..704)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             758..1417
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(824..843)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1759..2064
                     /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     1943..1962
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2105..2229)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2279..2295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2279..2295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2292..2314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2303..2319
                     /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(2327..2333,2334..2351,2352..2357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2372..2393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2510..2527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2681..3269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2761..2780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3440..4255)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3440..4255)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4163..4182
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4256..4360)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4428..4446
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
       61 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
      121 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
      181 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
      241 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
      301 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
      361 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgacgcg tattgggatg
      421 gcgcgcctat cgaaattcac tatagggaga cccaagctgg ctagttaagc ttcgccacca
      481 tgggtaagcc tatccctaac cctctcctcg gtctcgattc tacgacaagt ttgtacaaaa
      541 aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
      601 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg cggccgcatt
      661 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta
      721 ggatccggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
      781 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      841 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      901 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      961 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1021 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1081 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1141 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1201 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1261 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1321 gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1381 acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg cgtggatccg gcttactaaa
     1441 agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga
     1501 tatgtatacc cgaagtatgt caaaaagagg tgtgctatga agcagcgtat tacagtgaca
     1561 gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg
     1621 taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa
     1681 atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg
     1741 agaacaggga ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat
     1801 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc
     1861 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1921 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1981 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     2041 aacctgatgt tctggggaat ataaatgtca ggctccgtta tacacagcca gtctgcaggt
     2101 cgaccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     2161 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcag ctttcttgta
     2221 caaagtggta gtaatgacct taggatccca atggcgcgcc gagcttggct cgagcatggt
     2281 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2341 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3421 agtaaacttg gtctgacagt tagaaaaact catcgagcat caaatgaaac tgcaatttat
     3481 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     3541 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     3601 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3661 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3721 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3781 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     3841 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3901 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttccca gggatcgcag
     3961 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4021 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4081 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4141 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     4201 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct
     4261 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4321 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4381 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4441 cgaggccctt t
//
