LOCUS       sequence-261518-        3974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human ETV6 gRNA-3 and 4 is a pair of gRNAs. Four ETV6 gRNA plasmids 
            used the same backbone(Addgene plasmid#41824). ETV6 gRNA-4 target 
            the second ETV6 exon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3974)
  AUTHORS   Wu M, Liu S, Gao Y, Bai H, Machairaki V, Li G, Chen T, Cheng L
  TITLE     Conditional gene knockout and reconstitution in human iPSCs with an 
            inducible Cas9 system.
  JOURNAL   Stem Cell Res. 2018 May;29:6-14. doi: 10.1016/j.scr.2018.03.003. 
            Epub 2018 Mar 10.
  PUBMED    29554589
REFERENCE   2  (bases 1 to 3974)
  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: "Stem Cell 
            Res."; date: "2018-05"; volume: "29:6-14. doi"; pages: " 
            10.1016/j.scr.2018.03.003. Epub 2018 Mar 10"
COMMENT     Sequence Label: human ETV6 gRNA-4
FEATURES             Location/Qualifiers
     source          1..3974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        30..105
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(117..141)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(226..244)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(227..244)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(262..278)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(262..278)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(275..297)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    286..302
                     /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(310..316,317..334,335..340))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    355..376
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(493..510)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(664..1252)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(744..763)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1390..1764)
                     /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      1761..1770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1970..2764)
                     /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(1970..2764)
                     /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(2081..2100)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2691..2710
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3113..3415)
                     /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(3215..3234)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3538..3560
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3552..3569
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3553..3569
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3576..3595
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3576..3594
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(3734..3974,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3734..3754
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3905..3924
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgc gggtcacgaa cactcacgcg ttttagagct agaaatagca agttaaaata
       61 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc
      121 agctttcttg tacaaagttg gcattaaagg gcgaattcca gcacactggc ggccgttact
      181 agtggatccg agctcggtac caagcttgat gcatagcttg agtattctat agtgtcacct
      241 aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
      301 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
      361 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
      421 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
      481 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
      541 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      601 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      661 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      721 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      781 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      841 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      901 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      961 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1021 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1081 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     1141 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     1201 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1261 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     1321 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1381 tagcacgtgt cagtcctgct cctcggccac gaagtgcacg cagttgccgg ccgggtcgcg
     1441 cagggcgaac tcccgccccc acggctgctc gccgatctcg gtcatggccg gcccggaggc
     1501 gtcccggaag ttcgtggaca cgacctccga ccactcggcg tacagctcgt ccaggccgcg
     1561 cacccacacc caggccaggg tgttgtccgg caccacctgg tcctggaccg cgctgatgaa
     1621 cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt cccgggagaa
     1681 cccgagccgg tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg tgagcaccgg
     1741 aacggcactg gtcaacttgg ccatggtggc cctcctcacg tgctattatt gaagcattta
     1801 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1861 aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca
     1921 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact
     1981 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     2041 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     2101 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     2161 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     2221 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     2281 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     2341 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     2401 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     2461 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     2521 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     2581 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     2641 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     2701 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     2761 tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga
     2821 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
     2881 gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc
     2941 gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc
     3001 agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct
     3061 acgtgaaaag gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc
     3121 cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca
     3181 cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
     3241 tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac
     3301 gtgcactggc cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta
     3361 catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta
     3421 cgtataggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3481 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     3541 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     3601 tgggccctct agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc
     3661 cctttgtaca aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa
     3721 ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     3781 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     3841 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     3901 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     3961 cttgtggaaa ggac
//
