LOCUS Exported 6377 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION C-terminal tagging with mAID-mCherry2 using CRISPR/Cas9. ACCESSION . VERSION . KEYWORDS pMK293 (mAID-mCherry2-Hygro) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6377) AUTHORS Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT TITLE Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. JOURNAL Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001. Epub 2016 Mar 24. PUBMED 27052166 REFERENCE 2 (bases 1 to 6377) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6377 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 4..459 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(91..110) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 301..322 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 681..884 /codon_start=1 /product="minimal degron from the auxin-responsive IAA17 protein of Arabidopsis thaliana (Kubota et al., 2013)" /label=mini-AID /note="acts as an auxin-inducible degron (AID) in the presence of TIR1" /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA AAFVKVSMDGAPYLRKIDLRMYK" CDS 891..1601 /codon_start=1 /product="derivative of the monomeric red fluorescent protein mCherry" /label=mCherry2 /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1036..1054) /label=mCherry-R /note="mCherry, reverse primer" polyA_signal 1719..1840 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1756..1775) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1810..1829 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind complement(1871..1904) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1965..2464 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2000..2023) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 2397..2416 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 2525..3544 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE" protein_bind complement(4107..4140) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(4189..4209) /label=T3 /note="T3 promoter, forward primer" promoter complement(4189..4207) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4228..4244) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4228..4244) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4241..4263) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4252..4268 /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(4276..4306) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4321..4342 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4459..4476) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4630..5218) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4710..4729) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5389..6249) /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" primer_bind 6012..6031 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6250..6354) /gene="bla" /label=AmpR promoter ORIGIN 1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc 661 cgggatccgg tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc 721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg 781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg 841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaagctagc atggtgagca 901 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg 961 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg 1021 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg 1081 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca 1141 tccccgacta cttgaagctg tccttccccg agggcttcaa ttgggagcgc gtgatgaact 1201 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca 1261 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagtgtc 1321 gtaccatggg ctgggaggcc tccactgagc ggatgtaccc cgaggacggc gccctgaagg 1381 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga 1441 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc gacatcaagt 1501 tggacatcct ttcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg 1561 gccgccactc caccggcggc atggacgagc tgtacaagta atctagataa ctgatcataa 1621 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 1681 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 1741 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 1801 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtcgat catattcaat 1861 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag 1921 tttacgtcca gccaagctta ggatctcgac ctcgaaattc taccgggtag gggaggcgct 1981 tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac 2041 aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac cgactccgtt 2101 ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc 2161 cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt 2221 gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata 2281 gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg 2341 gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt 2401 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct 2461 ttcgacctgc atccatctag atctcgagca gctgaagctt accgctagca tggatagatc 2521 cggaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 2581 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 2641 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 2701 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 2761 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 2821 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 2881 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 2941 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 3001 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 3061 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 3121 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 3181 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 3241 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 3301 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 3361 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 3421 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 3481 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 3541 ataggctagc cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca 3601 tgcatcgatg atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga 3661 tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta 3721 cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa 3781 atgcctgctc tttactgaag gctctttact attgctttat gataatgttt catagttgga 3841 tatcataatt taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat 3901 ctatagatct atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt 3961 tctaagtact gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc 4021 ctctgttcca catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa 4081 gctggtcgag atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat 4141 taggtccctc gaagaggttc actaggatcc ggtacccagc ttttgttccc tttagtgagg 4201 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4261 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4321 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4381 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4441 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4501 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4561 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 4861 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5041 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 5101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 5401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 5581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 5641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 5881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 5941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 6121 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 6181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6241 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6301 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6361 tccccgaaaa gtgccac //