LOCUS       Exported                8472 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor HOXC13.
ACCESSION   .
VERSION     .
KEYWORDS    pHOXC13.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8472)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8472)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8472
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        169..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          698..925
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      937..946
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             946..1011
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1018..1038
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1063..5163
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5164..5211
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5239..5256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5245..5452
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5461..5601
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5461..5590
                     /label=AAV2 ITR
     rep_origin      5676..6131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5763..5782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5973..5994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6148..6167)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6267..6289
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6327..6345)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6413..6517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6518..7378
                     /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(6736..6755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7549..8137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8038..8057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8199..8439
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8199..8219
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8370..8389
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8469..72
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
       61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact
      181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      541 attttgtgca gcgatggggg cggggggggg ggggcggggc ggggcgaggc ggagaggtgc
      601 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
      661 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
      721 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      781 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      841 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      901 cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac
      961 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
     1021 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
     1081 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     1141 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     1201 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     1261 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     1321 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     1381 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     1441 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     1501 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     1561 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     1621 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     1681 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     1741 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     1801 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     1861 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     1921 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     1981 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     2041 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     2101 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     2161 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     2221 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     2281 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     2341 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     2401 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     2461 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     2521 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     2581 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     2641 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     2701 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     2761 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     2821 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     2881 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     2941 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     3001 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     3061 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     3121 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     3181 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     3241 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     3301 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     3361 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     3421 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     3481 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     3541 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     3601 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     3661 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     3721 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     3781 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     3841 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     3901 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     3961 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     4021 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     4081 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     4141 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     4201 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     4261 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     4321 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     4381 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     4441 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     4501 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     4561 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     4621 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     4681 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     4741 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     4801 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     4861 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     4921 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     4981 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     5041 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     5101 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     5161 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc
     5221 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     5281 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     5341 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     5401 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     5461 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     5521 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     5581 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     5641 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5701 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5761 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5821 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5881 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5941 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6001 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     6061 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     6121 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6181 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6241 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6301 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     6361 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     6421 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6481 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6541 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6601 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6661 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6721 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6781 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6841 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6901 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6961 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     7021 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     7081 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7141 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7201 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     7261 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7321 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7381 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7441 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7501 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7561 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7621 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7681 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7741 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7801 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7861 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7921 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7981 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     8041 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     8101 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     8161 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc
     8221 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     8281 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     8341 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     8401 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca agcagcgggt
     8461 gggtggtcgt tt
//
