LOCUS Exported 6329 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Provides the repair template with PP2Ac microhomology on each end for PP2Ac N-ter FAID tagging. ACCESSION . VERSION . KEYWORDS pN-PITCh-FAID SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6329) AUTHORS Lin DW, Chung BP, Huang JW, Wang X, Huang L, Kaiser P TITLE Microhomology based CRISPR tagging tools for protein tracking, purification, and depletion. JOURNAL J Biol Chem. 2019 May 28. pii: RA119.008422. doi: 10.1074/jbc.RA119.008422. PUBMED 31138654 REFERENCE 2 (bases 1 to 6329) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6329 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 86..674 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 575..594 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 828..845 /label=L4440 /note="L4440 vector, forward primer" primer_bind 943..960 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 944..960 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 1416..1432 /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)." regulatory 1587..1596 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1593..2309 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1638..1659) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1899..1918) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2246..2267 /label=EGFP-C /note="EGFP, forward primer" CDS 2319..2372 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 2373..2969 /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(2373..2392) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2869..2889 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 2979..3035 /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 3039..3062 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3063..3266 /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(3344..3361) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3350..3461 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3579..3599) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3584..3913 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3764..3899 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3826..3845 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3962..4987 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 5117..5238 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5154..5173) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5208..5227 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(5291..6151) /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 5914..5933 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6152..6243) /gene="bla" /label=AmpR promoter primer_bind 6285..6304 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6310..6329 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc 61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc 301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc 901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt 961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg 1021 taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag 1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg 1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata 1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa 1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa 1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt 1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc 1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 1501 ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt 1561 gtttggggcg ggtggcatca tggacggcca ccatggtgag caagggcgag gagctgttca 1621 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1681 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 1741 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 1801 agtgcttcag ccgttacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1861 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1921 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1981 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 2041 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 2101 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 2161 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 2221 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 2281 tcactctcgg catggacgag ctgtacaagg gaagcggaga gggcagagga agtctgctaa 2341 catgcggtga cgtcgaggag aatcctggac ctatgaccga gtacaagccc acggtgcgcc 2401 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact 2461 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc 2521 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 2581 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 2641 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 2701 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 2761 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 2821 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct 2881 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca 2941 cctggtgcat gacccgcaag cccggtgccg gaagcggagc tactaacttc agcctgctga 3001 agcaggctgg cgacgtggag gagaaccctg gacctatgga ctacaaagac gatgacgaca 3061 agaaggagaa gagtgcttgt cctaaagatc cagccaaacc tccggccaag gcacaagttg 3121 tgggatggcc accggtgaga tcataccgga agaacgtgat ggtttcctgc caaaaatcaa 3181 gcggtggccc ggaggcggcg gcgttcgtga aggtatcaat ggacggagca ccgtacttga 3241 ggaaaatcga tttgaggatg tataaagacg agaaggtgtt caccaaggag ccaaacacgt 3301 acgcgtacga tgctctagaa tgctgatggg ctagcaaaat cagcctcgac tgtgccttct 3361 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 3421 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct 3481 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 3541 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 3601 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3661 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 3721 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 3781 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 3841 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 3901 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt 3961 gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga 4021 cagcgtttcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga 4081 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga 4141 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat 4201 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt 4261 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga 4321 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg 4381 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta 4441 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga 4501 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg 4561 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc 4621 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc 4681 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc 4741 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga 4801 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg 4861 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg 4921 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa 4981 ggaatagcac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 5041 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 5101 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag 5161 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 5221 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta 5281 atcattgtca ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5341 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5401 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat 5461 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 5521 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5581 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta 5641 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 5701 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5761 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 5821 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5881 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5941 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6001 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6061 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6121 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca 6181 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6241 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta 6301 tacctgaata tggctcataa caccccttg //