LOCUS       Exported                8487 bp ds-DNA     circular SYN 02-MAY-2019
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    pX330_Cdk13_intron4_sg4
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8487)
  AUTHORS   Sara Dubbury
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, May 2, 2019 from SnapGene 3.3.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8487
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_RNA        271..346
                     /label=g RNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
                     /note="color: #ffffff"
     promoter        730..986
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
                     /note="color: #ffffff; direction: RIGHT"
     intron          987..1214
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /note="color: #------"
     regulatory      1226..1235
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             1235..1300
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 1300"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1307..1327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             1352..5452
                     /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"
                     /note="color: #993366"
                     /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             5453..5500
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5534..5741
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   5750..5890
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     repeat_region   5750..5879
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     rep_origin      5965..6420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        6702..6806
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             6807..7667
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6807 .. 6875 / #ccffcc / signal sequence
                        2: 6876 .. 7667 / #ccffcc
                     Cleavage site after base 6875"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7838..8426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
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 tgtatcacca gccctcaaga gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      901 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      961 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
     1021 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
     1081 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
     1141 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
     1201 tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta
     1261 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
     1321 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     1381 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     1441 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     1501 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     1561 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     1621 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     1681 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     1741 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     1801 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     1861 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     1921 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     1981 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     2041 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     2101 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     2161 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     2221 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     2281 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     2341 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     2401 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     2461 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     2521 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     2581 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     2641 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     2701 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     2761 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     2821 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     2881 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     2941 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     3001 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     3061 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     3121 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     3181 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     3241 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     3301 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     3361 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     3421 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     3481 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     3541 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     3601 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     3661 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     3721 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     3781 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     3841 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     3901 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     3961 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     4021 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     4081 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     4141 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     4201 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     4261 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     4321 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     4381 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     4441 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     4501 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     4561 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     4621 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     4681 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     4741 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     4801 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     4861 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     4921 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     4981 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     5041 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     5101 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     5161 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     5221 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     5281 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     5341 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     5401 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     5461 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc
     5521 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5581 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     5641 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     5701 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     5761 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     5821 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     5881 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5941 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     6001 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     6061 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     6121 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     6181 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     6241 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     6301 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     6361 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     6421 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     6481 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     6541 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6601 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     6661 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6721 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6781 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6841 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6901 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6961 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     7021 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     7081 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     7141 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     7201 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     7261 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     7321 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     7381 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     7441 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     7501 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     7561 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     7621 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     7681 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     7741 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7801 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7861 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7921 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7981 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     8041 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     8101 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     8161 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     8221 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     8281 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     8341 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     8401 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     8461 ccttttgctg gccttttgct cacatgt
//
