LOCUS       sequence-173348-        3971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a gRNA targeted to human TAZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3971)
  AUTHORS   Wang G, Yang L, Grishin D, Rios X, Ye LY, Hu Y, Li K, Zhang D, 
            Church GM, Pu WT
  TITLE     Efficient, footprint-free human iPSC genome editing by consolidation
            of Cas9/CRISPR and piggyBac technologies.
  JOURNAL   Nat Protoc. 2017 Jan;12(1):88-103. doi: 10.1038/nprot.2016.152. Epub
            2016 Dec 8.
  PUBMED    27929521
REFERENCE   2  (bases 1 to 3971)
  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"; doi: 
            "10.1038/nprot.2016.152"; journalName: "Nat Protoc"; date: 
            "2017-01"; volume: "12"; issue: "1"; pages: "88-103"
COMMENT     Sequence Label: pU6-gRNA-TAZ
FEATURES             Location/Qualifiers
     source          1..3971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(357..381)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(455..474)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(456..474)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(481..498)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(481..497)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(490..512)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             635..937
                     /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     816..835
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1286..2080
                     /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             1286..2080
                     /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(1340..1359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1950..1969
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2280..2289
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2286..2660
                     /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"
     rep_origin      2798..3386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3287..3306
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3540..3557
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3674..3695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3710..3715,3716..3733,3734..3740)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3748..3764
                     /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     3753..3775
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3772..3788
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3772..3788
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3806..3824
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3806..3823
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    3896..3920
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg aagctcaacc atggggactg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc
      361 agctttcttg tacaaagttg gcattaaggg cgaattctgc agatatccat cacactggcg
      421 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc
      481 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
      541 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
      601 cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag
      661 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg
      721 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
      781 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
      841 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
      901 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa
      961 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc
     1021 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
     1081 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
     1141 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
     1201 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca
     1261 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     1321 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     1381 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     1441 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     1501 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     1561 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     1621 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     1681 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     1741 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     1801 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     1861 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     1921 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     1981 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     2041 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt
     2101 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     2161 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2221 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgaggaggg
     2281 ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     2341 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     2401 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     2461 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     2521 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     2581 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     2641 tggccgagga gcaggactga cacgtgctaa aacttcattt ttaatttaaa aggatctagg
     2701 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2761 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2821 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2881 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2941 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3001 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3061 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3121 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3181 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3241 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3301 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3361 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3421 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3481 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3541 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3601 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3661 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     3721 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     3781 gctatgacca tgattacgcc aagctattta ggtgacacta tagaatactc aagctatgca
     3841 tcaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattcgcc
     3901 tttgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg
     3961 tcgggcagga a
//
