LOCUS       Exported                6242 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CDS of dCas9 with B3 nomenclature.
ACCESSION   .
VERSION     .
KEYWORDS    pUPD2 dCas9 (GB2664)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6242)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 6242)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6242
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             25..45
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             46..4146
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     terminator      4199..4256
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(4280..4297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4451..5039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4531..4550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5222..5316
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5337..5996)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5911..5930
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     6158..6176
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      6180..6223
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    6226..5
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 tagagcgatg agtggtctcg aatgcccaag aagaagagga aggtggacaa gaagtactcc
       61 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag
      121 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac
      181 ctcattggcg ccctcctgtt cgactccggg gaaacggccg aagccacgcg gctcaaaaga
      241 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt
      301 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg
      361 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg
      421 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact
      481 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga
      541 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc
      601 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt
      661 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc
      721 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc
      781 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg
      841 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac
      901 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg
      961 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat
     1021 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag
     1081 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga
     1141 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc
     1201 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac
     1261 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa
     1321 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt
     1381 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact
     1441 cgcaaatcag aagagactat cactccctgg aacttcgagg aagtcgtgga taagggggcc
     1501 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag
     1561 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag
     1621 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa
     1681 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa
     1741 gattatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc
     1801 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc
     1861 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt
     1921 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa
     1981 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg
     2041 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat
     2101 ggatttgcca accggaactt catgcagttg atccatgatg actctctcac ctttaaggag
     2161 gacatccaga aagcacaagt ttctggccag ggggacagtc tccacgagca catcgctaat
     2221 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa
     2281 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag
     2341 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag
     2401 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt
     2461 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag
     2521 gaactggaca tcaatcggct ctccgactac gacgtggatg ccatcgtgcc ccagtctttt
     2581 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag
     2641 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg
     2701 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt
     2761 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag
     2821 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat
     2881 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ttcagatttc
     2941 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat
     3001 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct
     3061 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag
     3121 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc
     3181 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac
     3241 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc
     3301 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc
     3361 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg
     3421 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg
     3481 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc
     3541 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga
     3601 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt
     3661 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg
     3721 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa
     3781 ggatctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt
     3841 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac
     3901 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca
     3961 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac
     4021 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca
     4081 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt
     4141 ggagacagca gggctgcagc ctgagaccac tcatcgcgac tagtagcggc cgctgcagtc
     4201 cggcaaaaaa gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac
     4261 ttcgcgttat gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4321 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4381 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4441 tgctggcgtt tttccacagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4501 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4561 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4621 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4681 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4741 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4801 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4861 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4921 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4981 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5041 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5101 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5161 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagc tcgaggcttg
     5221 gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga
     5281 gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat atcaaattac
     5341 gccccgccct gccactcgtc gcagtactgt tgtaattcat taagcattct gccgacatgg
     5401 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     5461 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     5521 tttaaatcaa aactggtgaa actcacccag ggattggctg atacgaaaaa catattctca
     5581 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     5641 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagtgatga aaacgtttca
     5701 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     5761 tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     5821 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     5881 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     5941 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     6001 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     6061 tcattatggt gaaagttgga acctcttacg tgcccgatca actcgagtgc cacctgacgt
     6121 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggcagaa
     6181 tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggaatt cgcggccgct
     6241 tc
//
