LOCUS       sequence-311465-        6569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human RAD51(T309A) mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6569)
  AUTHORS   Marzio A, Puccini J, Kwon Y, Maverakis NK, Arbini A, Sung P, 
            Bar-Sagi D, Pagano M
  TITLE     The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi 
            Sensitivity in Triple-Negative Breast Cancers.
  JOURNAL   Mol Cell. 2019 Jan 17;73(2):224-237.e6. doi: 
            10.1016/j.molcel.2018.11.003. Epub 2018 Dec 13.
  PUBMED    30554948
REFERENCE   2  (bases 1 to 6569)
  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"; doi: 
            "10.1016/j.molcel.2018.11.003"; journalName: "Mol Cell"; date: 
            "2019-01-17- 17"; volume: "73"; issue: "2"; pages: "224-237.e6"
COMMENT     Sequence Label: pcDNA3.1 2xFLAG-2xSTREP RAD51 (T309A)
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             923..946
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             962..985
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1001..1024
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1061..1084
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2160..2177)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2166..2390
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2436..2864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2523..2542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2733..2754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2873..2893)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2878..3207
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3058..3193
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3120..3139
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3274..4068
                     /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(3328..3347)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3938..3957
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4242..4363
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4279..4298)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4333..4352
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4412..4428)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4412..4428)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4425..4447)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4436..4452
                     /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(4460..4466,4467..4484,4485..4490))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4505..4526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4643..4660)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4814..5402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4894..4913)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5573..6364,6365..6433))
                     /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     6196..6215
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6434..6538)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttacca tggactacaa ggacgacgat gacaaggtac caggatctat
      961 ggattataaa gatgatgatg ataaagggtc ggccgccagc tggagccacc ctcagttcga
     1021 gaagggagga ggaagcggcg gaggcagcgg aggaggaagc tggagccacc cgcagttcga
     1081 gaaaggagct ggatctaata tggcaatgca gatgcagctt gaagcaaatg cagatacttc
     1141 agtggaagaa gaaagctttg gcccacaacc catttcacgg ttagagcagt gtggcataaa
     1201 tgccaacgat gtgaagaaat tggaagaagc tggattccat actgtggagg ctgttgccta
     1261 tgcgccaaag aaggagctaa taaatattaa gggaattagt gaagccaaag ctgataaaat
     1321 tctggctgag gcagctaaat tagttccaat gggtttcacc actgcaactg aattccacca
     1381 aaggcggtca gagatcatac agattactac tggctccaaa gagcttgaca aactacttca
     1441 aggtggaatt gagactggat ctatcacaga aatgtttgga gaattccgaa ctgggaagac
     1501 ccagatctgt catacgctag ctgtcacctg ccagcttccc attgaccggg gtggaggtga
     1561 aggaaaggcc atgtacattg acactgaggg tacctttagg ccagaacggc tgctggcagt
     1621 ggctgagagg tatggtctct ctggcagtga tgtcctggat aatgtagcat atgctcgagc
     1681 gttcaacaca gaccaccaga cccagctcct ttatcaagca tcagccatga tggtagaatc
     1741 taggtatgca ctgcttattg tagacagtgc caccgccctt tacagaacag actactcggg
     1801 tcgaggtgag ctttcagcca ggcagatgca cttggccagg tttctgcgga tgcttctgcg
     1861 actcgctgat gagtttggtg tagcagtggt aatcactaat caggtggtag ctcaagtgga
     1921 tggagcagcg atgtttgctg ctgatcccaa aaaacctatt ggaggaaata tcatcgccca
     1981 tgcatcaaca accagattgt atctgaggaa aggaagaggg gaagccagaa tctgcaaaat
     2041 ctacgactct ccctgtcttc ctgaagctga agctatgttc gccattaatg cagatggagt
     2101 gggagatgcc aaagactgaa ttgtcgagtc tagagggccc gtttaaaccc gctgatcagc
     2161 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2221 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2281 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2341 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2401 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2461 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2521 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2581 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2641 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2701 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2761 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2821 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2881 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2941 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3001 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3061 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3121 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3181 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3241 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3301 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3361 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3421 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3481 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3541 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3601 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3661 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3721 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3781 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3841 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3901 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3961 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4021 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4081 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4141 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4201 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4261 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4321 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4381 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4441 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4501 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4561 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4621 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4681 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4741 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4801 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4861 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4921 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4981 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5041 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5101 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5161 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5221 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5281 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5341 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5401 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5461 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5521 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5581 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5641 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5701 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5761 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5821 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5881 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5941 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6001 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6061 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6121 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6181 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6241 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6301 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6361 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6421 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6481 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6541 catttccccg aaaagtgcca cctgacgtc
//
