LOCUS       sequence-287976-        3988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains tracrRNA-hU6 sequence for double sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3988)
  AUTHORS   Xie Y, Wang D, Lan F, Wei G, Ni T, Chai R, Liu D, Hu S, Li M, Li D, 
            Wang H, Wang Y
  TITLE     An episomal vector-based CRISPR/Cas9 system for highly efficient 
            gene knockout in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2017 May 24;7(1):2320. doi: 10.1038/s41598-017-02456-y.
  PUBMED    28539611
REFERENCE   2  (bases 1 to 3988)
  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: "2017-05-24"; volume: "7(1):2320. doi"; pages: " 
            10.1038/s41598-017-02456-y"
COMMENT     Sequence Label: gRNAU6
FEATURES             Location/Qualifiers
     source          1..3988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        57..132
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        174..422
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     174..194
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     345..364
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        443..518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(530..554)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(639..657)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(640..657)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(675..691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(675..691)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(688..710)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    699..715
                     /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(723..729,730..747,748..753))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    768..789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(906..923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1077..1665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1157..1176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1803..2177)
                     /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"
     regulatory      2174..2183
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2383..3177)
                     /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             complement(2383..3177)
                     /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(2494..2513)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3104..3123
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3526..3828)
                     /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     complement(3628..3647)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3951..3973
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3965..3982
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3966..3982
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggc cctctagatg catgctcgag ctcttcgttt
       61 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      121 ccgagtcggt gctttttttg tcgactggat ccggtaccaa ggtcgggcag gaagagggcc
      181 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
      241 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
      301 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
      361 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
      421 ccgtgagacg taactcgtct cggttttaga gctagaaata gcaagttaaa ataaggctag
      481 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctaga cccagctttc
      541 ttgtacaaag ttggcattaa agggcgaatt ccagcacact ggcggccgtt actagtggat
      601 ccgagctcgg taccaagctt gatgcatagc ttgagtattc tatagtgtca cctaaatagc
      661 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      721 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      781 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      841 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      901 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      961 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1441 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1501 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1561 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1621 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1681 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1741 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttagcacg
     1801 tgtcagtcct gctcctcggc cacgaagtgc acgcagttgc cggccgggtc gcgcagggcg
     1861 aactcccgcc cccacggctg ctcgccgatc tcggtcatgg ccggcccgga ggcgtcccgg
     1921 aagttcgtgg acacgacctc cgaccactcg gcgtacagct cgtccaggcc gcgcacccac
     1981 acccaggcca gggtgttgtc cggcaccacc tggtcctgga ccgcgctgat gaacagggtc
     2041 acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga agtcccggga gaacccgagc
     2101 cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg cggtgagcac cggaacggca
     2161 ctggtcaact tggccatggt ggccctcctc acgtgctatt attgaagcat ttatcagggt
     2221 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2281 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     2341 aaggagaaaa taccgcatca ggaaattgta agcgttaata attcagaaga actcgtcaag
     2401 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     2461 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     2521 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2581 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     2641 gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2701 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2761 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2821 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2881 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2941 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     3001 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     3061 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     3121 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     3181 aaacgatcct catcctgtct cttgatcaga gcttgatccc ctgcgccatc agatccttgg
     3241 cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc
     3301 agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt
     3361 aagcccactg caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc
     3421 ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgaa
     3481 aaggatctag gtgaagatcc tttttgataa tctcatgcct gacatttata ttccccagaa
     3541 catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc
     3601 cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc
     3661 cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact
     3721 ggccaggggg atcaccatcc gtcgccccgg cgtgtcaata atatcactct gtacatccac
     3781 aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgcc gtacgtatag
     3841 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     3901 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     3961 acgttgtaaa acgacggcca gtgaattg
//
