LOCUS       sequence-193873-        3973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AAVS1 sgRNA. Target sequence: GGGGCCACTAGGGACAGGAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  AUTHORS   Rose JC, Stephany JJ, Valente WJ, Trevillian BM, Dang HV, Bielas JH,
            Maly DJ, Fowler DM
  TITLE     Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics.
  JOURNAL   Nat Methods. 2017 Jul 24. doi: 10.1038/nmeth.4368.
  PUBMED    28737741
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: "Nat 
            Methods. 2017 Jul 24. doi: 10.1038/nmeth.4368."
COMMENT     Sequence Label: AAVS1_sgRNA
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(692..1066)
                     /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      1063..1072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1272..2066)
                     /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(1272..2066)
                     /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(1383..1402)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1993..2012
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2415..2717)
                     /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(2517..2536)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     2840..2862
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2854..2871
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2855..2871
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2878..2897
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2878..2896
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3036..3284
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3036..3056
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3207..3226
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3305..3380
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3392..3416)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3501..3519)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3502..3519)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3537..3553)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3537..3553)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3550..3572)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3561..3577
                     /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(3585..3591,3592..3609,3610..3615))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3630..3651
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3768..3785)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3939..3973,1..554))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
       61 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      121 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      181 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      241 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      301 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      361 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      421 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      481 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      541 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      601 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      661 ttttaaatta aaaatgaagt tttagcacgt gtcagtcctg ctcctcggcc acgaagtgca
      721 cgcagttgcc ggccgggtcg cgcagggcga actcccgccc ccacggctgc tcgccgatct
      781 cggtcatggc cggcccggag gcgtcccgga agttcgtgga cacgacctcc gaccactcgg
      841 cgtacagctc gtccaggccg cgcacccaca cccaggccag ggtgttgtcc ggcaccacct
      901 ggtcctggac cgcgctgatg aacagggtca cgtcgtcccg gaccacaccg gcgaagtcgt
      961 cctccacgaa gtcccgggag aacccgagcc ggtcggtcca gaactcgacc gctccggcga
     1021 cgtcgcgcgc ggtgagcacc ggaacggcac tggtcaactt ggccatggtg gccctcctca
     1081 cgtgctatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     1141 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     1201 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa
     1261 gcgttaataa ttcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     1321 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     1381 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     1441 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     1501 ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt
     1561 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     1621 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     1681 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     1741 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     1801 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     1861 cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg
     1921 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     1981 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     2041 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagag
     2101 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     2161 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccat
     2221 aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg ctttctcttt
     2281 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     2341 cgtttctgcg gactggcttt ctacgtgaaa aggatctagg tgaagatcct ttttgataat
     2401 ctcatgcctg acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt
     2461 tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata
     2521 tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg
     2581 gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgccccggc
     2641 gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag
     2701 gtgtaaacct taaactgccg tacgtatagg ctgcgcaact gttgggaagg gcgatcggtg
     2761 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     2821 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa
     2881 tacgactcac tatagggcga attgggccct ctagatgcat gctcgagcgg ccgccagtgt
     2941 gatggatatc tgcagaattc gccctttgta caaaaaagca ggctttaaag gaaccaattc
     3001 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     3061 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     3121 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3181 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     3241 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggcc actagggaca
     3301 ggatgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     3361 aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa agttggcatt
     3421 aaagggcgaa ttccagcaca ctggcggccg ttactagtgg atccgagctc ggtaccaagc
     3481 ttgatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     3541 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3601 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3661 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3721 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3781 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3841 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3901 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3961 gacgagcatc aca
//
