LOCUS       sequence-261524-        3973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human ETV6 gRNA-3 is a 20-nt gRNA expression plasmid targeting the 
            second ETV6 exon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  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 3973)
  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-3
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        41..116
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(128..152)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(237..255)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(238..255)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(273..289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(273..289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(286..308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    297..313
                     /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(321..327,328..345,346..351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    366..387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(504..521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(675..1263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(755..774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1401..1775)
                     /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      1772..1781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1981..2775)
                     /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(1981..2775)
                     /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(2092..2111)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2702..2721
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3124..3426)
                     /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(3226..3245)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3549..3571
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3563..3580
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3564..3580
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3587..3606
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3587..3605
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(3745..3973,1..20)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3745..3765
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3916..3935
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tgtggaaagg acgaaacacc ggcgtcgagg aagcgtaact gttttagagc tagaaatagc
       61 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      121 tttctagacc cagctttctt gtacaaagtt ggcattaaag ggcgaattcc agcacactgg
      181 cggccgttac tagtggatcc gagctcggta ccaagcttga tgcatagctt gagtattcta
      241 tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      301 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      361 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
      421 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      481 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      541 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      601 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      661 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      721 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      781 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      841 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      901 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      961 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1021 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1081 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     1141 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1201 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1261 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1321 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1381 aaatgaagtt ttagcacgtg tcagtcctgc tcctcggcca cgaagtgcac gcagttgccg
     1441 gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc
     1501 ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg
     1561 tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc
     1621 gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag
     1681 tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg
     1741 gtgagcaccg gaacggcact ggtcaacttg gccatggtgg ccctcctcac gtgctattat
     1801 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1861 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     1921 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaataat
     1981 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     2041 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     2101 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     2161 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     2221 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     2281 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     2341 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     2401 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     2461 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     2521 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     2581 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     2641 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     2701 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     2761 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagagc ttgatcccct
     2821 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac
     2881 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca
     2941 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt
     3001 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg
     3061 actggctttc tacgtgaaaa ggatctaggt gaagatcctt tttgataatc tcatgcctga
     3121 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct
     3181 gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat
     3241 catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc
     3301 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgccccggcg tgtcaataat
     3361 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt
     3421 aaactgccgt acgtataggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3481 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3541 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     3601 atagggcgaa ttgggccctc tagatgcatg ctcgagcggc cgccagtgtg atggatatct
     3661 gcagaattcg ccctttgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga
     3721 tccggtacca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
     3781 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     3841 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     3901 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     3961 gctttatata tct
//
