LOCUS Exported 5394 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION C-terminal tagging with mAID using CRISPR/Cas9. ACCESSION . VERSION . KEYWORDS pMK286 (mAID-NeoR) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5394) 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 5394) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5394 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 159..179 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter complement(204..308) /gene="bla" /label=AmpR promoter rep_origin 335..790 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" polyA_signal 797..918 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(808..827) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 862..881 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(1039..1242) /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" primer_bind complement(1278..1297) /label=T7 /note="T7 promoter, forward primer" promoter complement(1279..1297) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1307..1324) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1307..1323) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1316..1338) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(1464..1919) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 1946..2050 /gene="bla" /label=AmpR promoter CDS 2051..2911 /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 complement(2269..2288) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3082..3670 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3571..3590 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3824..3841 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3958..3979 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3994..4024 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4032..4048 /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)." primer_bind 4037..4059 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4056..4072 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4056..4072 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4091..4111 /label=T3 /note="T3 promoter, forward primer" promoter 4093..4111 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 4203..4222 /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" CDS complement(4410..5204) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4521..4540) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 5131..5150 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(5239..202) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5253..5388 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(5307..5326) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" ORIGIN 1 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc 61 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg 121 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca 181 cagctggttc tttccgcctc aggactcttc ctttttcaat attattgaag catttatcag 241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 301 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc 361 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 421 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 481 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 541 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 601 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 661 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 721 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 781 cttacaattt acgcgttaag atacattgat gagtttggac aaaccacaac tagaatgcag 841 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 901 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 961 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat 1021 gatcagttat ctagattatt tatacatcct caaatcgatt ttcctcaagt acggtgctcc 1081 gtccattgat accttcacga acgccgccgc ctccgggcca ccgcttgatt tttggcagga 1141 aaccatcacg ttcttccggt atgatctcac cggtggccat cccacaactt gtgccttggc 1201 cggaggtttg gctggatctt taggacaagc actcttctcc ttggcgcctg caccggatcc 1261 cggggagctc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 1321 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 1381 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 1441 tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt 1501 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 1561 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 1621 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 1681 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 1741 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 1801 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 1861 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 1921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 1981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 2041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 2101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 2161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 2221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 2281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 2341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 2401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 2461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 2521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 2581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 2641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 2701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 2821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 2881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 2941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 3001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3841 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 3901 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 3961 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 4021 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 4081 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctgggtaccg gatccaacga 4141 cccaacaccg tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg 4201 tattgtctcc ttccgtgttt cagttagcct ccccctaggg tgggcgaaga actccagcat 4261 gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa 4321 cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg 4381 gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag 4441 gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat 4501 tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc 4561 gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata 4621 ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc 4681 ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc 4741 tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg 4801 tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg 4861 atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg 4921 cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga 4981 acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca 5041 ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg 5101 gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc 5161 caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat 5221 cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt 5281 ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca 5341 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcgg //