LOCUS Exported 5124 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Transient expression of humanized MS2 (V75E/A81G), fused to mVenus in mammalian cells, under a hUBC promoter, V5 tag. ACCESSION . VERSION . KEYWORDS pUBC_V5_MS2-mVenus SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5124) AUTHORS Maass PG, Barutcu AR, Shechner DM, Weiner CL, Mele M, Rinn JL TITLE Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING). JOURNAL Nat Struct Mol Biol. 2018 Jan 8. pii: 10.1038/s41594-017-0015-3. doi: 10.1038/s41594-017-0015-3. PUBMED 29343869 REFERENCE 2 (bases 1 to 5124) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5124 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 618..637 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(856..960) /gene="bla" /label=AmpR promoter primer_bind 1050..1069 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1263..1285 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 1277..1294 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1278..1294 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2371..2390 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 2552..2561 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2564..2584 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2591..2632 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 3062..3781 /codon_start=1 /product="Venus YFP with monomerizing A206K mutation (Nagai et al., 2002; Kremers et al., 2006)" /label=mVenus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3107..3128) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3368..3387) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3715..3736 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3828..3847) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3882..3901 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3958..3974) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3958..3974) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3971..3993) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3982..3998 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4006..4036) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4051..4072 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4189..4206) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4360..4948) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4440..4459) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5119..855) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" ORIGIN 1 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 61 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 121 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 181 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 241 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 301 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 361 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 421 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 481 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 541 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 601 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 661 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 721 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 781 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 841 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 901 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 961 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 1021 ctataaaaat aggcgtatca cgagctatgc ggcatcagag cagattgtac tgagagtgca 1081 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca 1141 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 1201 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 1261 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cacgtatgtc 1321 gaggtaggcg cgccgggttt tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc 1381 tgccacgtca gacgaagggc gcagcgagcg tcctgatcct tccgcccgga cgctcaggac 1441 agcggcccgc tgctcataag actcggcctt agaaccccag tatcagcaga aggacatttt 1501 aggacgggac ttgggtgact ctagggcact ggttttcttt ccagagagcg gaacaggcga 1561 ggaaaagtag tcccttctcg gcgattctgc ggagggatct ccgtggggcg gtgaacgccg 1621 atgattatat aaggacgcgc cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg 1681 gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg 1741 ctggccgggg ctttcgtggc cgccgggccg ctcggtggga cggaagcgtg tggagagacc 1801 gccaagggct gtagtctggg tccgcgagca aggttgccct gaactggggg ttggggggag 1861 cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag acgcttgtga ggcgggctgt 1921 gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc 1981 ttcgctaatg cgggaaagct cttattcggg tgagatgggc tggggcacca tctggggacc 2041 ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt cgtctgttgc gggggcggca 2101 gttatggcgg tgccgttggg cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg 2161 tcgtgacgtc acccgttctg ttggcttata atgcagggtg gggccacctg ccggtaggtg 2221 tgcggtaggc ttttctccgt cgcaggacgc agggttcggg cctagggtag gctctcctga 2281 atcgacaggc gccggacctc tggtgagggg agggataagt gaggcgtcag tttctttggt 2341 cggttttatg tacctatctt cttaagtagc tgaagctccg gttttgaact atgcgctcgg 2401 ggttggcgag tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt 2461 caatatgtaa ttttcagtgt tagactagta aattgtccgc taaattctgg ccgtttttgg 2521 cttttttgtt agacctggag gtaccgccac cgccaccatg gctccaaaga agaagcgtaa 2581 ggtaggcagc ggcaagccca tccccaaccc cctgctgggc ctggacagca ccggcagcgc 2641 tagcaatttc actcaatttg tgctcgtgga taacggagga acgggcgacg tgaccgtggc 2701 accaagcaac tttgctaatg gagtggccga gtggattagc tcaaatagca ggtcccaggc 2761 ttacaaggtc acctgtagtg tccggcaatc atccgcccag aatcggaaat atactattaa 2821 ggtagaagtg ccgaaagtgg caactcaaac cgtggggggc gaggaattgc ctgtggccgg 2881 ctggagaagc tacctcaaca tggaactcac cattccaatc ttcgccacaa acagcgactg 2941 tgagctcatc gtcaaagcca tgcagggtct gctgaaggac ggcaatccta ttccatctgc 3001 aatcgcggcc aattccggca tttatggatc caagggctcg ggctcgacct cgggctccgg 3061 aatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 3121 cggcgacgta aacggacaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 3181 cggcaagctg accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac 3241 cctcgtgacc accctgggat acggcctgca gtgcttcgcc cgctaccccg accacatgaa 3301 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 3361 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 3421 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 3481 caagctggag tacaactaca acagccacaa cgtctatatc accgccgaca agcagaagaa 3541 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc 3601 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 3661 ctacctgagc taccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt 3721 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta 3781 atgataagtc gacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 3841 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 3901 caatgtatct tattcgactg tttaaacctg caggcatgca agcttggcgt aatcatggtc 3961 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 4021 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 4081 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 4141 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4201 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4261 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 4321 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4381 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4441 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4501 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 4561 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4621 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4681 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4741 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4801 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4861 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4921 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4981 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5041 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5101 gtaaacttgg tctgacagtt acca //