LOCUS Exported 4202 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION NLSPUFb pCR8 gateway donor vector for insertion of N-terminal domain (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest. ACCESSION . VERSION . KEYWORDS pAC1406-pCR8-4xNLS_PUFb_2xNLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4202) 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 4202) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4202 /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" protein_bind complement(2081..2180) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(2197..2216) /label=T7 /note="T7 promoter, forward primer" promoter complement(2198..2216) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2221..2237) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2221..2237) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 2668..3459 /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 3552..4140 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4041..4060 /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 atggctgccg tgttatccag 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 gttatgtaat 1561 cgaacatgta ctggagcacg gtcgtcctga ggataaaagc aaaattgtag cagaaatccg 1621 aggcaatgta cttgtattga gtcagcacaa atttgcaaac aatgttgtgc 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 cctgattaat taagaattcg acccagcttt cttgtacaaa 2101 gttggcatta taaaaaataa ttgctcatca atttgttgca acgaacaggt cactatcagt 2161 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg 2221 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca 2281 caagataaaa atatatcatc atgcctcctc tagaccagcc aggacagaaa tgcctcgact 2341 tcgctgctgc ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc 2401 cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc 2461 aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg 2521 gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg 2581 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc 2641 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact 2701 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta 2761 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg 2821 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg 2881 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt 2941 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa 3001 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg 3061 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag 3121 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg 3181 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc 3241 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg 3301 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat 3361 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac 3421 tacgtgaaag gcgagatcac caaggtagtc ggcaaataac cctcgagcca cccatgacca 3481 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc 3541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 3601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 3661 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 3721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 3781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 3841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3901 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg 3961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 4021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 4081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 4141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 4201 tt //