LOCUS       sequence-313968-        8111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9-based dual-fluorescent DSB repair reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8111)
  AUTHORS   Eki R, She J, Parlak M, Benamar M, Du KP, Kumar P, Abbas T
  TITLE     A robust CRISPR-Cas9-based fluorescent reporter assay for the 
            detection and quantification of DNA double-strand break repair.
  JOURNAL   Nucleic Acids Res. 2020 Oct 17. pii: 5929239. doi: 
            10.1093/nar/gkaa897.
  PUBMED    33068408
REFERENCE   2  (bases 1 to 8111)
  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: "Nucleic 
            Acids Res. 2020 Oct 17. pii: 5929239. doi: 10.1093/nar/gkaa897."
COMMENT     Sequence Label: lenti-CDDR
FEATURES             Location/Qualifiers
     source          1..8111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      215..224
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             542..595
                     /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             608..1315
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(750..768)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1018..1037
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1709..2209
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2128..2146
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2221..2616
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(2680..2700)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3098..3109)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3314..3547
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3619..3740
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3656..3675)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3710..3729
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3780..3915
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3915..3986
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3987..4055,4056..4847)
                     /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     complement(4205..4224)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5018..5606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5507..5526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5760..5777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5894..5915
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5930..5935,5936..5953,5954..5960)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5968..5984
                     /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)."
     primer_bind     5973..5995
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5992..6008
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5992..6008
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6027..6047
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6029..6047
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6075..6301
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6302..6482
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6529..6654
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7147..7380
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7565..7609
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7907..8024
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggagccacc atggagagcg acgagagcgg
      241 cctgcccgcc atggagatcg agtgccgcat caccggcacc ctgaacggcg tggagttcga
      301 gctggtgggc ggcggagagg gcacccccga gcagggccgc atgaccaaca agatgaagag
      361 caccaaaggc gccctgacct tcagccccta cctgctgagc cacgtgatgg gctacggctt
      421 ctaccacttc ggcacctacc ccagcggcta cgagaacccc ttcctgcacg ccatcaacaa
      481 cggcggctac accaacaccc gcatcgagaa gtacgaggac ggcggcgtgc tgcagtggga
      541 tgagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt
      601 cgccaccgtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
      661 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      721 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      781 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      841 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      901 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      961 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     1021 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     1081 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     1141 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1201 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1261 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaccctg
     1321 aacgtgagct tcagctaccg ctacgaggcc ggccgcgtga tcggcgactt caaggtgatg
     1381 ggcaccggct tccccgagga cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc
     1441 accgtggagc acctgcaccc catgggcgat aacgatctgg atggcagctt cacccgcacc
     1501 ttcagcctgc gcgacggcgg ctactacagc tccgtggtgg acagccacat gcacttcaag
     1561 agcgccatcc accccagcat cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg
     1621 gaggaggatc acagcaacac cgagctgggc atcgtggagt accagcacgc cttcaagacc
     1681 ccggatgcag atgccggtga agaataactt gcgccttttc caaggcagcc ctgggtttgc
     1741 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc
     1801 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
     1861 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
     1921 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
     1981 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
     2041 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
     2101 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
     2161 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg gggtaccacc
     2221 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     2281 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     2341 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     2401 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     2461 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     2521 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     2581 cgtgaattgc tgccctctgg ttatgtgtgg gagggcctgc agctgcagta acccgggtaa
     2641 ctaagtaagg atcgatccaa gatatcgtat tcttaactat gttgctcctt ttacgctatg
     2701 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2761 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     2821 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     2881 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     2941 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3001 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     3061 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3121 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3181 gacgagtcgg atctcccttt gggccgcctc cccgcctgtt tcgcctcggc gtccggacta
     3241 gaggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     3301 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     3361 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     3421 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     3481 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     3541 tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     3601 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3661 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3721 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     3781 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3841 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3901 gcttttttgg aggcctttca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3961 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4021 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4081 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4141 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4201 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     4261 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4321 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4381 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4441 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4501 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4561 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4621 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4681 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4741 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4801 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4861 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4921 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4981 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5041 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5101 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5161 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5221 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5281 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5341 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5401 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5461 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5521 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5581 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5641 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5701 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5761 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     5821 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5881 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5941 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     6001 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     6061 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     6121 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     6181 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     6241 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     6301 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     6361 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     6421 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     6481 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     6541 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     6601 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     6661 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     6721 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     6781 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     6841 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     6901 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     6961 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     7021 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     7081 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     7141 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     7201 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     7261 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     7321 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     7381 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     7441 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     7501 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     7561 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     7621 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     7681 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     7741 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     7801 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     7861 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     7921 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     7981 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     8041 acagcagaga tccagtttgg ttactagcta ggtcttgaaa ggagtgggaa ttggctccgg
     8101 tgcccgtcag t
//
