LOCUS Exported 6491 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION 4xNLS_PUFa_2xNLS_mCBPHAT in pCR8 Gateway donor vector. ACCESSION . VERSION . KEYWORDS pAC1415-pCR8-4xNLS_PUFa_2xNLS_mCBPHAT 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" CDS 719..739 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 743..763 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 767..787 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 878..898 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1955..1975 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1979..1999 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" regulatory 3680..3689 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 catgatcgat ggcggaggcg gatctgatcc 721 aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtagatccaa aaaagaagag 781 aaaggtagga tctaccggat ctagaaacga tggtggtggt ggaagcgggg gtgggggcag 841 cggtggaggg ggaagcgggc gcgccgggat cctccccccc aagaaaaaga ggaaggtatc 901 tagaggccgc agccgccttt tggaagattt tcgaaacaac cggtacccca atttacaact 961 gcgggagatt gctggacata taatggaatt ttcccaagac cagcatgggt ccagattcat 1021 tcagctgaaa ctggagcgtg ccacaccagc tgagcgccag cttgtcttca atgaaatcct 1081 ccaggctgcc taccaactca tggtggatgt gtttggtaat tacgtcattc agaagttctt 1141 tgaatttggc agtcttgaac agaagctggc tttggcagaa cggattcgag gccacgtcct 1201 gtcattggca ctacagatgt atggcagccg tgttatcgag aaagctcttg agtttattcc 1261 ttcagaccag cagaatgaga tggttcggga actagatggc catgtcttga agtgtgtgaa 1321 agatcagaat ggcaatcacg tggttcagaa atgcattgaa tgtgtacagc cccagtcttt 1381 gcaatttatc atcgacgcgt ttaagggaca ggtatttgcc ttatccacac atccttatgg 1441 ctgccgagtg attcagagaa tcctggagca ctgtctccct gaccagacac tccctatttt 1501 agaggagctt caccagcaca cagagcagct ggtacaggat caatatggaa attatgtaat 1561 ccaacatgta ctggagcacg gtcgtcctga ggataaaagc aaaattgtag cagaaatccg 1621 aggcaatgta cttgtattga gtcagcacaa atttgcaagc aatgttgtgg agaagtgtgt 1681 tactcacgcc tcacgtacgg agcgcgctgt gctcatcgac gaggtgtgca ccatgaacga 1741 cggtccccac agtgccttat acaccatgat gaaggaccag tatgccaact acgtggtcca 1801 gaagatgatt gacgtggcgg agccaggcca gcggaagatc gtcatgcata agatccggcc 1861 ccacatcgca actcttcgta agtacaccta tggcaagcac attctggcca agctggagaa 1921 gtactacatg aagaacggtg ttgacttagg ggacccaaag aagaagcgca aagtggatcc 1981 taaaaagaaa agaaaggtag gcggccgcgg gggaggcggt tccggtggcg gcggaagcgg 2041 aggtggagga tcagggccgg ccatctttaa acccgaggag ctacgccagg cacttatgcc 2101 aactctagaa gcactctatc gacaggaccc agagtctttg ccttttcgtc agcctgtaga 2161 tcctcagctc ctaggaatcc cagattattt tgatatagtg aagaatccta tggacctttc 2221 taccatcaaa cgaaagctgg acacagggca atatcaagaa ccctggcagt atgtggatga 2281 tgtctggctt atgttcaaca atgcgtggct atataatcgt aaaacgtccc gtgtatataa 2341 attttgcagt aaacttgcag aggtctttga acaagaaatt gaccctgtca tgcagtctct 2401 tggatattgc tgtggacgaa agtatgagtt ctccccacag actttgtgct gttacggaaa 2461 gcagctgtgt acaattcctc gtgatgcagc ctactacagc tatcagaata ggtatcattt 2521 ctgtgagaag tgtttcacag agatccaggg cgagaatgtg accctgggtg acgacccttc 2581 ccaacctcag acgacaattt ccaaggatca atttgaaaag aagaaaaatg ataccttaga 2641 tcctgaacct tttgttgact gcaaagagtg tggccggaag atgcatcaga tttgtgttct 2701 acactatgac atcatttggc cttcaggttt tgtgtgtgac aactgtttga agaaaactgg 2761 cagacctcgg aaagaaaaca aattcagtgc taagaggctg cagaccacac gattgggaaa 2821 ccacttagaa gacagagtga ataagttttt gcggcgccag aatcaccctg aagctgggga 2881 ggtttttgtc agagtggtgg ccagctcaga caagactgtg gaggtcaagc cgggaatgaa 2941 gtcaaggttt gtggattctg gagagatgtc ggaatctttc ccatatcgta ccaaagcact 3001 ctttgctttt gaggagatcg atggagtcga tgtgtgcttt tttgggatgc atgtgcaaga 3061 atacggctct gattgccccc caccaaatac aaggcgtgta tacatatctt atctggacag 3121 tattcatttc ttccggcccc gctgcctccg gacagctgtt taccatgaga tcctcatcgg 3181 atatctcgag tatgtgaaga aattggggta tgtgacagga catatttggg cctgtccccc 3241 aagtgaagga gatgactata tctttcattg ccacccccct gaccagaaaa tccccaaacc 3301 aaaacgacta caggagtggt acaagaagat gctggacaag gcgtttgcag agaggatcat 3361 taacgactat aaggacatct tcaaacaagc gaacgaagac aggctcacga gtgccaagga 3421 gttgccctat tttgaaggag atttctggcc taatgtgttg gaagaaagca ttaaggaact 3481 agaacaagaa gaagaagaaa ggaaaaaaga agagagtact gcagcgagtg agactcctga 3541 gggcagtcag ggtgacagca aaaatgcgaa gaaaaagaac aacaagaaga ccaacaaaaa 3601 caaaagcagc attagccgcg ccaacaagaa gaagcccagc atgcccaatg tttccaacga 3661 cctgtcgcag aagctgtatg ccaccatgga gaagcacaag gaggtattct ttgtgattca 3721 tctgcatgct gggcctgtta tcagcactca gccccccatc gtggaccctg atcctctgct 3781 tagctgtgac ctcatggatg ggcgagatgc cttcctcacc ctggccagag acaagcactg 3841 ggaattctct tccttacgcc gctccaaatg gtccactctg tgcatgctgg tggagctgca 3901 cacacagggc caggaccgct ttgtttatac ctgcaatgag tgcaaacacc atgtggaaac 3961 acgctggcac tgcactgtgt gtgaggacta tgacctttgt atcaattgct acaacacaaa 4021 gagccacacc cataagatgg tgaagtgggg gctaggccta gatgatgagg gcagcagtca 4081 gggtgagcca cagtccaaga gcccccagga atcccggcgt ctcagcatcc agcgctgcat 4141 ccagtccctg gtgcatgcct gccagtgtcg caatgccaac tgctcactgc cgtcttgcca 4201 gaagatgaag cgagtcgtgc agcacaccaa gggctgcaag cgcaagacta atggaggatg 4261 cccagtgtgc aagcagctca ttgctctttg ctgctaccac gccaaacact gccaagaaaa 4321 taaatgccct gtgcccttct gcctcaacat ctagttaatt 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 //