LOCUS       sequence-192071-        8484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a sgRNA and a Cas9 EQR variant that recognizes ""NGAG"" 
            PAM motifs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8484)
  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 8484)
  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..8484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..983
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          984..1211
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1223..1232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1232..1297
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1304..1324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1349..3064
                     /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             5450..5497
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5525..5542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5531..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5747..5887
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5747..5876
                     /label=AAV2 ITR
     rep_origin      5962..6417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6049..6068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6259..6280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6434..6453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6553..6575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6613..6631)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6699..6803
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6804..6872,6873..7664)
                     /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(7022..7041)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7835..8423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8324..8343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      901 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      961 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1021 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1081 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     1141 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     1201 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     1261 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1321 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1381 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1441 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1501 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1561 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1621 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1681 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1741 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1801 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1861 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1921 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1981 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2041 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     2101 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     2161 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     2221 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2281 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2341 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2401 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2461 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2521 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2581 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2641 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2701 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2761 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2821 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2881 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2941 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3001 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3061 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     3121 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     3181 cgaggacatt ctggaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat
     3241 tgaggaaaga ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa
     3301 gaggcgtcgc tatacgggct ggggacgatt gtcgcggaaa cttatcaacg ggataagaga
     3361 caagcaaagt ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa
     3421 ctttatgcag ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca
     3481 ggtttccgga caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc
     3541 catcaaaaag ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg
     3601 acgtcacaaa ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa
     3661 ggggcaaaaa aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg
     3721 cagccagatc ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta
     3781 cctctattac ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg
     3841 tttatctgat tacgacgtcg atcacattgt accccaatcc tttttgaagg acgattcaat
     3901 cgacaataaa gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag
     3961 cgaggaagtc gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat
     4021 aacgcaaaga aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga
     4081 caaggccgga tttattaaac gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc
     4141 acagatacta gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga
     4201 agtcaaagta atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt
     4261 ctataaagtt agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt
     4321 cgtagggacc gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga
     4381 ttacaaagtt tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc
     4441 tacagccaaa tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct
     4501 ggcaaacgga gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat
     4561 cgtatgggat aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt
     4621 caacatagta aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc
     4681 aaaaaggaat agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg
     4741 tggcttcgag agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg
     4801 aaaatccaag aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc
     4861 gtcttttgaa aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa
     4921 ggatctcata attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg
     4981 gatgttggct agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata
     5041 cgtgaatttc ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa
     5101 cgaacagaag caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca
     5161 aatttcggaa ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag
     5221 cgcatacaac aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt
     5281 gtttactctt accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga
     5341 tcgcaaacag tacagatcta ccaaggaggt gctagacgcg acactgattc accaatccat
     5401 cacgggatta tatgaaactc ggatagattt gtcacagctt gggggtgaca aaaggccggc
     5461 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga
     5521 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5581 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5641 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5701 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg
     5761 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     5821 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     5881 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5941 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     6001 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     6061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     6121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     6181 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     6241 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     6301 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     6361 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     6421 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     6481 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     6541 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     6601 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     6661 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6721 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6781 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6841 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6901 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6961 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     7021 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     7081 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     7141 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     7201 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     7261 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     7321 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     7381 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     7441 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     7501 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     7561 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     7621 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7681 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7741 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7801 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     7861 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     7921 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7981 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     8041 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     8101 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     8161 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     8221 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     8281 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     8341 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     8401 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     8461 tttgctggcc ttttgctcac atgt
//
