LOCUS       sequence-266392-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a loss of function mutant of an engineered variant of 
            RAD18 (""e18"").
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-07-30"; volume: "10(1):3395. doi"; pages: " 
            10.1038/s41467-019-11105-z"
COMMENT     Sequence Label: pcDNA3.1(+)- e18 LOF
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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(join(4583..4589,4590..4607,4608..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(join(5696..6487,6488..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 atttagccag
     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
//
