LOCUS       sequence-313974-        4130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter-less BFP cDNA with two homology arms to the lenti-CDDR 
            reporter for HDR detection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4130)
  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 4130)
  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: BFP Homology Donor
FEATURES             Location/Qualifiers
     source          1..4130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             360..413
                     /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             426..1124
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     primer_bind     complement(1544..1563)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1545..1563)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1570..1587)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1570..1586)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1579..1601)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1714..1818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1819..1887,1888..2679)
                     /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(2037..2056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2850..3438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3339..3358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3500..3748
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3500..3520
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3671..3690
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3773..3848
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    4033..4054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4069..4074,4075..4092,4093..4099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4107..4123
                     /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     join(4112..4130,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcttggtac catggagagc gacgagagcg
       61 gcctgcccgc catggagatc gagtgccgca tcaccggcac cctgaacggc gtggagttcg
      121 agctggtggg cggcggagag ggcacccccg agcagggccg catgaccaac aagatgaaga
      181 gcaccaaagg cgccctgacc ttcagcccct acctgctgag ccacgtgatg ggctacggct
      241 tctaccactt cggcacctac cccagcggct acgagaaccc cttcctgcac gccatcaaca
      301 acggcggcta caccaacacc cgcatcgaga agtacgagga cggcggcgtg ctgcagtatg
      361 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaccggtcg
      421 ccaccagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
      481 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
      541 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
      601 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
      661 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
      721 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
      781 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
      841 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
      901 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
      961 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
     1021 aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca
     1081 gatactgcga cctccctagc aaactggggc acaagcttaa ttaatgaacg tgagcttcag
     1141 ctaccgctac gaggccggcc gcgtgatcgg cgacttcaag gtgatgggca ccggcttccc
     1201 cgaggacagc gtgatcttca ccgacaagat catccgcagc aacgccaccg tggagcacct
     1261 gcaccccatg ggcgataacg atctggatgg cagcttcacc cgcaccttca gcctgcgcga
     1321 cggcggctac tacagctccg tggtggacag ccacatgcac ttcaagagcg ccatccaccc
     1381 cagcatcctg cagaacgggg gccccatgtt cgccttccgc cgcgtggagg aggatcacag
     1441 caacaccgag ctgggcatcg tggagtacca gcacgccttc aagaccccgg atgcagatgc
     1501 cggtgaagaa taactcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
     1561 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1621 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1681 caccggtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1741 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1801 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1861 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1921 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1981 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2041 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2101 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2161 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2221 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2281 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2341 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2401 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2461 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2521 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2581 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2641 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2701 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2761 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3481 tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     3541 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     3601 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     3661 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     3721 atatatcttg tggaaaggac gaaacaccgg aagcaggtga gaacctgcct ctgttttaga
     3781 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     3841 gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt
     3901 tagcgcgtgc gccaattcag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     3961 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4021 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     4081 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
//
