LOCUS       sequence-326712-        3974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector to target the SOX2 stop codon for HDR gene 
            tagging in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3974)
  AUTHORS   Abatti LE, Lado-Fernandez P, Huynh L, Collado M, Hoffman MM, 
            Mitchell JA
  TITLE     Epigenetic reprogramming of a distal developmental enhancer cluster 
            drives SOX2 overexpression in breast and lung adenocarcinoma.
  JOURNAL   Nucleic Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734.
  PUBMED    37738673
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: "Nucleic 
            Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734."
COMMENT     Sequence Label: gRNA_SOX2_HDR
FEATURES             Location/Qualifiers
     source          1..3974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    342..366
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        complement(378..453)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(475..723)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(533..552)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(703..723)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(862..881)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(863..881)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(888..905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(888..904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(897..919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1042..1344
                     /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     1223..1242
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1693..2487
                     /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             1693..2487
                     /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(1747..1766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2357..2376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2687..2696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2693..3067
                     /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      3205..3793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3694..3713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3947..3964
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gccctttaat gccaactttg tacaagaaag
      361 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
      421 cttattttaa cttgctattt ctagctctaa aacccctcca gttcgctgtc cggcggtgtt
      481 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
      541 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
      601 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
      661 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
      721 ctcttcctgc ccgaccttgg taccggatcc agtcgactga attggttcct ttaaagcctg
      781 cttttttgta caaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat
      841 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      901 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      961 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     1021 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
     1081 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
     1141 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1201 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1261 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1321 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
     1381 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     1441 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     1501 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     1561 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     1621 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
     1681 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     1741 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1801 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1861 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1921 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1981 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2041 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2101 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2161 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2221 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2281 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2341 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2401 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2461 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     2521 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     2581 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2641 gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca ccatggccaa
     2701 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
     2761 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
     2821 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
     2881 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
     2941 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
     3001 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     3061 ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3121 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3181 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3241 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3301 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3361 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3421 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3481 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3541 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3601 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3661 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3721 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3781 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3841 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3901 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3961 cgaggaagcg gaag
//
