LOCUS       sequence-195312-        8324 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses a sgRNA and a Cas9 EQR variant that recognizes ""NGAG"" 
            PAM motifs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8324)
  AUTHORS   Lambrus BG, Moyer TC, Holland AJ
  TITLE     Applying the auxin-inducible degradation system for rapid protein 
            depletion in mammalian cells.
  JOURNAL   Methods Cell Biol. 2018;144:107-135. doi: 
            10.1016/bs.mcb.2018.03.004. Epub 2018 Apr 5.
  PUBMED    29804665
REFERENCE   2  (bases 1 to 8324)
  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: "Methods 
            Cell Biol."; date: "2018"; volume: "144:107-135. doi"; pages: " 
            10.1016/bs.mcb.2018.03.004. Epub 2018 Apr 5"
COMMENT     Sequence Label: px330 EQR
FEATURES             Location/Qualifiers
     source          1..8324
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      226..235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             235..300
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             307..327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             352..2067
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4453..4500
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4528..4545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4534..4741
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4750..4890
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4750..4879
                     /label=AAV2 ITR
     rep_origin      4965..5420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5052..5071)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5262..5283
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5437..5456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5556..5578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5616..5634)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5702..5806
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5807..5875,5876..6667)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6025..6044)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6838..7426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7327..7346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7488..7736
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7488..7508
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7659..7678
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7755..7830
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        7927..8212
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
       61 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      121 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
      181 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat
      241 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag
      301 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
      361 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
      421 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
      481 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
      541 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
      601 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
      661 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
      721 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
      781 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
      841 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
      901 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
      961 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     1021 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     1081 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     1141 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     1201 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     1261 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     1321 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     1381 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     1441 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     1501 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     1561 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     1621 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     1681 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     1741 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     1801 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     1861 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     1921 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     1981 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     2041 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     2101 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     2161 gacttcctgg acaatgagga aaacgaggac attctggaag atatagtgtt gactcttacc
     2221 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac
     2281 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg
     2341 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag
     2401 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc
     2461 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt
     2521 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg
     2581 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca
     2641 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata
     2701 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc
     2761 caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt
     2821 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa
     2881 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga
     2941 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg
     3001 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag
     3061 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc
     3121 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac
     3181 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg
     3241 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg
     3301 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta
     3361 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa
     3421 agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat
     3481 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa
     3541 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga
     3601 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg
     3661 ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag
     3721 gactgggacc cgaaaaagta cggtggcttc gagagcccta cagttgccta ttctgtccta
     3781 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg
     3841 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg
     3901 aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt
     3961 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac
     4021 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag
     4081 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat
     4141 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat
     4201 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag
     4261 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc
     4321 aagtattttg acacaacgat agatcgcaaa cagtacagat ctaccaagga ggtgctagac
     4381 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag
     4441 cttgggggtg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     4501 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     4561 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     4621 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     4681 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     4741 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4801 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4861 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4921 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4981 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5041 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5101 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5161 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5221 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5281 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5341 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5401 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5461 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5521 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5581 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5641 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5701 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5761 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5821 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5881 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5941 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6001 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6061 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6121 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6181 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6241 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6301 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6361 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6421 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6481 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     6541 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6601 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6661 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6721 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6781 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6841 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6901 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6961 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     7021 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7081 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7141 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7201 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7261 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7321 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7381 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7441 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtgag ggcctatttc
     7501 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     7561 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     7621 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     7681 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgggt
     7741 cttcgagaag acctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     7801 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt
     7861 taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctctaga
     7921 ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     7981 cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     8041 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     8101 tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg
     8161 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
     8221 gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta
     8281 tttatttatt ttttaattat tttgtgcagc gatgggggcg gggg
//
