LOCUS Exported 3396 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Donor backbone vector to clone left and right HR arms in for Degron-KI application. ACCESSION . VERSION . KEYWORDS Degron-KI donor backbone SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3396) AUTHORS Zhou Q, Derti A, Ruddy DA, Rakiec DP, Kao I, Lira M, Gibaja V, Chan H, Yang Y, Min J, Schlabach MR, Stegmeier F TITLE A chemical genetics approach for the functional assessment of novel cancer genes. JOURNAL Cancer Res. 2015 Mar 18. pii: canres.2930.2014. PUBMED 25788694 REFERENCE 2 (bases 1 to 3396) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3396 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 278..300 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 292..309 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 293..309 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 320..339 /label=T7 /note="T7 promoter, forward primer" promoter 320..338 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 343..354 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 431..1027 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(431..450) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 927..947 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1037..1093 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1094..1417 /codon_start=1 /product="destabilization domain that can be stabilized by Shield1 in the ProteoTuner(TM) system" /label=DD /note="L106P mutant of FKBP12" /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL LKPE" primer_bind complement(1602..1619) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1712..2300) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1792..1811) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2474..3268) /codon_start=1 /product="aminoglycoside phosphotransferase" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" promoter complement(3269..3373) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 361 aggcctaggc gcgccatgag ctcagcggaa gcagtccgca tatgggagtg gttgttaaga 421 gatgctcgag atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 481 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 541 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 601 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 661 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 721 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg 781 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa 841 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc 901 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 961 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 1021 cggtgccgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga 1081 gaaccctgga cctatgggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt 1141 ccccaagcgc ggccagacct gtgtggtgca ctacaccggg atgcttgaag atggaaagaa 1201 agtcgattcc tcccgggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt 1261 gatccgaggc tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac 1321 tatatctcca gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc 1381 cactctcgtc ttcgatgtgg agcttctaaa accggaaccg gaattctttt gaaaataagc 1441 tcctgagcgg ccgcggacga ctagctaacc gcggtacctc ttaattaact ggcctcatgg 1501 gccttccttt cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaacat 1561 ggtcatagct gtttccttgc gtattgggcg ctctccgctt cctcgctcac tgactcgctg 1621 cgctcggtcg ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag caaaaggcca 1681 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 1741 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 1801 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 1861 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 1921 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 1981 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2041 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2101 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 2161 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 2221 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 2281 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 2341 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 2401 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 2461 ggtctgacag ttattagaaa aattcatcca gcagacgata aaacgcaata cgctggctat 2521 ccggtgccgc aatgccatac agcaccagaa aacgatccgc ccattcgccg cccagttctt 2581 ccgcaatatc acgggtggcc agcgcaatat cctgataacg atccgccacg cccagacggc 2641 cgcaatcaat aaagccgcta aaacggccat tttccaccat aatgttcggc aggcacgcat 2701 caccatgggt caccaccaga tcttcgccat ccggcatgct cgctttcaga cgcgcaaaca 2761 gctctgccgg tgccaggccc tgatgttctt catccagatc atcctgatcc accaggcccg 2821 cttccatacg ggtacgcgca cgttcaatac gatgtttcgc ctgatgatca aacggacagg 2881 tcgccgggtc cagggtatgc agacgacgca tggcatccgc cataatgctc actttttctg 2941 ccggcgccag atggctagac agcagatcct gacccggcac ttcgcccagc agcagccaat 3001 cacggcccgc ttcggtcacc acatccagca ccgccgcaca cggaacaccg gtggtggcca 3061 gccagctcag acgcgccgct tcatcctgca gctcgttcag cgcaccgctc agatcggttt 3121 tcacaaacag caccggacga ccctgcgcgc tcagacgaaa caccgccgca tcagagcagc 3181 caatggtctg ctgcgcccaa tcatagccaa acagacgttc cacccacgct gccgggctac 3241 ccgcatgcag gccatcctgt tcaatcatac tcttcctttt tcaatattat tgaagcattt 3301 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 3361 taggggttcc gcgcacattt ccccgaaaag tgccac //