LOCUS Exported 6491 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mCBPHAT_4xNLS_PUFa_2xNLS in pCR8 Gateway donor vector. ACCESSION . VERSION . KEYWORDS pAC1416-pCR8-mCBPHAT_4xNLS_PUFa_2xNLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6491) AUTHORS Cheng AW, Jillette N, Lee P, Plaskon D, Fujiwara Y, Wang W, Taghbalout A, Wang H TITLE Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. JOURNAL Cell Res. 2016 Feb;26(2):254-7. doi: 10.1038/cr.2016.3. Epub 2016 Jan 15. PUBMED 26768771 REFERENCE 2 (bases 1 to 6491) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6491 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 387..473 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 522..544 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 536..553 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 537..553 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 569..668 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" regulatory 2312..2321 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3008..3028 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3032..3052 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3056..3076 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3167..3187 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4244..4264 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4268..4288 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind complement(4370..4469) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(4486..4505) /label=T7 /note="T7 promoter, forward primer" promoter complement(4487..4505) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4510..4526) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4510..4526) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 4957..5748 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 5841..6429 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6330..6349 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 661 agcaggctcc gaattcaccg gtgccgccac catgatcttt aaacccgagg agctacgcca 721 ggcacttatg ccaactctag aagcactcta tcgacaggac ccagagtctt tgccttttcg 781 tcagcctgta gatcctcagc tcctaggaat cccagattat tttgatatag tgaagaatcc 841 tatggacctt tctaccatca aacgaaagct ggacacaggg caatatcaag aaccctggca 901 gtatgtggat gatgtctggc ttatgttcaa caatgcgtgg ctatataatc gtaaaacgtc 961 ccgtgtatat aaattttgca gtaaacttgc agaggtcttt gaacaagaaa ttgaccctgt 1021 catgcagtct cttggatatt gctgtggacg aaagtatgag ttctccccac agactttgtg 1081 ctgttacgga aagcagctgt gtacaattcc tcgtgatgca gcctactaca gctatcagaa 1141 taggtatcat ttctgtgaga agtgtttcac agagatccag ggcgagaatg tgaccctggg 1201 tgacgaccct tcccaacctc agacgacaat ttccaaggat caatttgaaa agaagaaaaa 1261 tgatacctta gatcctgaac cttttgttga ctgcaaagag tgtggccgga agatgcatca 1321 gatttgtgtt ctacactatg acatcatttg gccttcaggt tttgtgtgtg acaactgttt 1381 gaagaaaact ggcagacctc ggaaagaaaa caaattcagt gctaagaggc tgcagaccac 1441 acgattggga aaccacttag aagacagagt gaataagttt ttgcggcgcc agaatcaccc 1501 tgaagctggg gaggtttttg tcagagtggt ggccagctca gacaagactg tggaggtcaa 1561 gccgggaatg aagtcaaggt ttgtggattc tggagagatg tcggaatctt tcccatatcg 1621 taccaaagca ctctttgctt ttgaggagat cgatggagtc gatgtgtgct tttttgggat 1681 gcatgtgcaa gaatacggct ctgattgccc cccaccaaat acaaggcgtg tatacatatc 1741 ttatctggac agtattcatt tcttccggcc ccgctgcctc cggacagctg tttaccatga 1801 gatcctcatc ggatatctcg agtatgtgaa gaaattgggg tatgtgacag gacatatttg 1861 ggcctgtccc ccaagtgaag gagatgacta tatctttcat tgccaccccc ctgaccagaa 1921 aatccccaaa ccaaaacgac tacaggagtg gtacaagaag atgctggaca aggcgtttgc 1981 agagaggatc attaacgact ataaggacat cttcaaacaa gcgaacgaag acaggctcac 2041 gagtgccaag gagttgccct attttgaagg agatttctgg cctaatgtgt tggaagaaag 2101 cattaaggaa ctagaacaag aagaagaaga aaggaaaaaa gaagagagta ctgcagcgag 2161 tgagactcct gagggcagtc agggtgacag caaaaatgcg aagaaaaaga acaacaagaa 2221 gaccaacaaa aacaaaagca gcattagccg cgccaacaag aagaagccca gcatgcccaa 2281 tgtttccaac gacctgtcgc agaagctgta tgccaccatg gagaagcaca aggaggtatt 2341 ctttgtgatt catctgcatg ctgggcctgt tatcagcact cagcccccca tcgtggaccc 2401 tgatcctctg cttagctgtg acctcatgga tgggcgagat gccttcctca ccctggccag 2461 agacaagcac tgggaattct cttccttacg ccgctccaaa tggtccactc tgtgcatgct 2521 ggtggagctg cacacacagg gccaggaccg ctttgtttat acctgcaatg agtgcaaaca 2581 ccatgtggaa acacgctggc actgcactgt gtgtgaggac tatgaccttt gtatcaattg 2641 ctacaacaca aagagccaca cccataagat ggtgaagtgg gggctaggcc tagatgatga 2701 gggcagcagt cagggtgagc cacagtccaa gagcccccag gaatcccggc gtctcagcat 2761 ccagcgctgc atccagtccc tggtgcatgc ctgccagtgt cgcaatgcca actgctcact 2821 gccgtcttgc cagaagatga agcgagtcgt gcagcacacc aagggctgca agcgcaagac 2881 taatggagga tgcccagtgt gcaagcagct cattgctctt tgctgctacc acgccaaaca 2941 ctgccaagaa aataaatgcc ctgtgccctt ctgcctcaac atcaacgatg gcggaggcgg 3001 atctgatcca aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tagatccaaa 3061 aaagaagaga aaggtaggat ctaccggatc tagaaacgat ggtggtggtg gaagcggggg 3121 tgggggcagc ggtggagggg gaagcgggcg cgccgggatc ctccccccca agaaaaagag 3181 gaaggtatct agaggccgca gccgcctttt ggaagatttt cgaaacaacc ggtaccccaa 3241 tttacaactg cgggagattg ctggacatat aatggaattt tcccaagacc agcatgggtc 3301 cagattcatt cagctgaaac tggagcgtgc cacaccagct gagcgccagc ttgtcttcaa 3361 tgaaatcctc caggctgcct accaactcat ggtggatgtg tttggtaatt acgtcattca 3421 gaagttcttt gaatttggca gtcttgaaca gaagctggct ttggcagaac ggattcgagg 3481 ccacgtcctg tcattggcac tacagatgta tggcagccgt gttatcgaga aagctcttga 3541 gtttattcct tcagaccagc agaatgagat ggttcgggaa ctagatggcc atgtcttgaa 3601 gtgtgtgaaa gatcagaatg gcaatcacgt ggttcagaaa tgcattgaat gtgtacagcc 3661 ccagtctttg caatttatca tcgacgcgtt taagggacag gtatttgcct tatccacaca 3721 tccttatggc tgccgagtga ttcagagaat cctggagcac tgtctccctg accagacact 3781 ccctatttta gaggagcttc accagcacac agagcagctg gtacaggatc aatatggaaa 3841 ttatgtaatc caacatgtac tggagcacgg tcgtcctgag gataaaagca aaattgtagc 3901 agaaatccga ggcaatgtac ttgtattgag tcagcacaaa tttgcaagca atgttgtgga 3961 gaagtgtgtt actcacgcct cacgtacgga gcgcgctgtg ctcatcgacg aggtgtgcac 4021 catgaacgac ggtccccaca gtgccttata caccatgatg aaggaccagt atgccaacta 4081 cgtggtccag aagatgattg acgtggcgga gccaggccag cggaagatcg tcatgcataa 4141 gatccggccc cacatcgcaa ctcttcgtaa gtacacctat ggcaagcaca ttctggccaa 4201 gctggagaag tactacatga agaacggtgt tgacttaggg gacccaaaga agaagcgcaa 4261 agtggatcct aaaaagaaaa gaaaggtagg cggccgcggg ggaggcggtt ccggtggcgg 4321 cggaagcgga ggtggaggat cagggccggc ctgattaatt aagaattcga cccagctttc 4381 ttgtacaaag ttggcattat aaaaaataat tgctcatcaa tttgttgcaa cgaacaggtc 4441 actatcagtc aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg 4501 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt 4561 tacattgcac aagataaaaa tatatcatca tgcctcctct agaccagcca ggacagaaat 4621 gcctcgactt cgctgctgcc caaggttgcc gggtgacgca caccgtggaa acggatgaag 4681 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg 4741 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg 4801 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag 4861 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg 4921 cagcagggca gtcgccctaa aacaaagtta aacatcatga gggaagcggt gatcgccgaa 4981 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg 5041 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt 5101 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac 5161 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc 5221 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa 5281 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac 5341 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca 5401 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa 5461 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt 5521 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct 5581 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga 5641 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa 5701 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac 5761 ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta 5821 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 5881 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 5941 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 6001 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 6061 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 6121 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 6181 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa 6241 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6301 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6361 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6421 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6481 gctcacatgt t //