LOCUS Exported 6902 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses an engineered variant of RAD18 (""e18"") that stimulates CRISPR mediated HDR.. ACCESSION . VERSION . KEYWORDS pcDNA3.1(+)- e18 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6902) AUTHORS Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, Huang JW, Leuzzi G, Cuella-Martin R, Palacios A, Gupta A, Egli D, Ciccia A TITLE Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. JOURNAL Nat Commun. 2019 Jul 30;10(1):3395. doi: 10.1038/s41467-019-11105-z. PUBMED 31363085 REFERENCE 2 (bases 1 to 6902) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6902 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 25..404 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 405..608 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 559..579 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 653..672 /label=T7 /note="T7 promoter, forward primer" promoter 653..671 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 725..734 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 734..757 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 770..796 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" protein_bind 812..836 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(2283..2300) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2289..2513 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2559..2987 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2646..2665) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2856..2877 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2996..3016) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3001..3330 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3181..3316 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3243..3262 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3397..4191 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3451..3470) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4061..4080 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4365..4486 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4402..4421) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4456..4475 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4535..4551) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4535..4551) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4548..4570) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4559..4575 /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(4583..4613) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4628..4649 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4766..4783) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4937..5525) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5017..5036) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5696..6556) /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 6319..6338 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6557..6661) /gene="bla" /label=AmpR promoter primer_bind complement(6736..6755) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 601 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga 661 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttggta ccgagctcgg 721 atccgccacc atggactaca aggatgacga tgacaagctc gatggaggat acccatacga 781 tgttccagat tacgctgctc gaggttatca aacaagtttg tacaaaaaag ttggcaggta 841 ctccctggcc gagtctcggt ggcctccggg cctggcagtc atgaagacaa tagatgattt 901 gctgcggtgt ggaatttgct tcgagtattt caacattgca atgataatac ctcagtgttc 961 acataactac tgctctctct gtataagaaa atttctgtcc tataaaactc agtgtccaac 1021 ttgctgtgtg actgtcacag agccggatct gaaaaataac cgcatattag atgaactggt 1081 aaaaagcttg aattttgcac ggaatcatct gctgcagttt gctttagagt caccagccaa 1141 atctcctgct tcttcctctt caaagaatct tgctgtcaaa gtatatactc ctgtagcctc 1201 cagacagtct ttaaagcagg ggagcaggtt aatggataat ttcttgatca gagaaatgag 1261 tggttctaca tcagagttgt tgataaaaga aaataaaagc aaattcagcc ctcaaaaaga 1321 ggcgagccct gctgcaaaga ccaaagagac acgttctgta gaagagatcg ctccagatcc 1381 ctcagaggct aagcgtcctg agccaccctc gacatccact ttgaaacaag ttactaaagt 1441 ggattgtcct gtttgcgggg ttaacattcc agaaagtcac attaataagc atttagacag 1501 ctgtttatca cgcgaagaga agaaggaaag cctcagaagt tctgttcaca aaaggaagcc 1561 gctgcccaaa actgtataca atgcccaatg cgatgctttg catcctaaat cagctgctga 1621 aatagttcaa gaaatcgaaa atatagagaa gactaggatg cgtcttgaag ctagtaaact 1681 caatgaaagt gtaatggttt ttacaaagga ccaaacagaa aaggaaatag atgaaatcca 1741 cagtaaatat cgtaaaaaac ataagagtga atttcagctt ctggtggatc aggctagaaa 1801 aggatacaag aaaattgctg gaatgtcaca aaaaacagta acaataacaa aagaagatga 1861 atctacagaa aagctatctt ctgtatgcat gggacaggaa gataatatga cctcagtaac 1921 aaaccacttt tctcaatcaa agctggactc cccagaggaa ttggaacctg acagagaaga 1981 ggattcttct agctgtattg atattcaaga agttctttct tcatcagaat cagattcatg 2041 caatagttcc agttcagaca tcataagaga tcttttagaa gaagaggaag cctgggaagc 2101 atcacataaa aacgatcttc aagacacaga aataagtcca agacagaatc gccgcacaag 2161 agccgctgaa agtgctgaga ttgaaccaag aaacaagcgt aaaagggatt aagaattctg 2221 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc 2281 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2341 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2401 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2461 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 2521 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 2581 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 2641 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 2701 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 2761 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 2821 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 2881 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 2941 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 3001 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 3061 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 3121 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 3181 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 3241 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga 3301 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc 3361 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac 3421 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 3481 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 3541 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 3601 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 3661 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 3721 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 3781 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 3841 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 3901 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 3961 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 4021 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 4081 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 4141 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 4201 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca 4261 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 4321 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag 4381 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 4441 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 4501 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 4561 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 4621 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 4681 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 4741 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 4801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 4861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 4921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 5041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 5101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 5161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 5221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 5281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcagt gctacagagt 5341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 5401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 5461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 5521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 5581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 5641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 5701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 5761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 5821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 5881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 5941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 6001 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 6061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 6121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 6181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 6241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 6301 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 6361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 6421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 6481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 6541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 6601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 6661 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct 6721 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc 6781 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca 6841 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg 6901 cg //