LOCUS       sequence-67842-e        7671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses K227M nickase variant of I-Ani I nuclease in mammalian 
            cells with BFP tracker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7671)
  AUTHORS   Kuhar R, Gwiazda KS, Humbert O, Mandt T, Pangallo J, Brault M, Khan 
            I, Maizels N, Rawlings DJ, Scharenberg AM, Certo MT
  TITLE     Novel fluorescent genome editing reporters for monitoring DNA repair
            pathway utilization at endonuclease-induced breaks.
  JOURNAL   Nucleic Acids Res. 2013 Oct 10.
  PUBMED    24121685
REFERENCE   2  (bases 1 to 7671)
  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. 2013 Oct 10."
COMMENT     Sequence Label: pCVL.SFFV.Kozak.HA.NLS.Y2AniK227M.IRES.BFP
FEATURES             Location/Qualifiers
     source          1..7671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..235
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    463..588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /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    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1955..2356
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2369..2378
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2381..2407
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2411..2431
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    join(3241..3815,3816..3818,3819..3827)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3408..3425)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3635..3654
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3844..4545
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    complement(5210..5243)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5448..5465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5454..5678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5751..5768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5922..6510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6002..6021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6681..7472,7473..7541))
                     /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     7304..7323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7542..7646)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
       61 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      121 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      181 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      481 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      541 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      601 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      661 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      721 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
      781 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
      841 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
      901 aaaagtaaga ccaccgcaca gcaagcggcc ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aatgaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttatcg attacgcgtc acgtgctagc tgcagtaacg ccattttgca aggcatggaa
     1981 aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac
     2041 gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca
     2101 gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg
     2161 cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg
     2221 aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc
     2281 gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct
     2341 ccgacagact gagtcgcccg ctcgagccgc caccatggga tatccatacg atgtcccaga
     2401 ttatgcgcca cctaagaaga aacgcaaagt cgaattcggg agcgatctga cctacgccta
     2461 tctggtgggc ctgtacgagg gcgacggata tttttccatc actaaaaagg gcaagtacct
     2521 gacctatgag ctgggaattg aactgtctat caaggatgtg cagctgatct acaagatcaa
     2581 gaagatcctg gggatcggca ttgtgagctt caggaagaga aacgagattg aaatggtggc
     2641 cctgaggatc agggataaga atcacctgaa atctaagatt ctgcccatct tcgagaagta
     2701 tcctatgttt agtaacaaac agtacgacta tctgaggttt agaaatgctc tgctgagcgg
     2761 catcatctac ctggaggatc tgccagacta tacccggtcc gacgagcccc tgaacagcat
     2821 cgaatccatc attaatacat cttacttcag tgcctggctg gtgggcttca tcgaggctga
     2881 agggtgcttc tctgtgtaca aactgaacaa ggacgatgac tatctgattg ccagttttga
     2941 tatcgctcag agggatggag acatcctgat tagcgccatc agaaagtacc tgtccttcac
     3001 cacaaaggtg tatctggaca aaacaaattg tagcaaactg aaggtcacta gcgtgcgctc
     3061 cgtggagaac atcattaagt tcctgcagaa tgctcctgtg aaactgctgg gcaacatgaa
     3121 gctgcagtac aaactgtggc tgaagcagct gcggaaaatc tctcgctaca gtgaaaaaat
     3181 caagattcca tccaattatt agcctgcagg tcgagcatgc atctagggcg gccaattccg
     3241 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     3301 tgcgtttgtc tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg
     3361 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     3421 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     3481 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     3541 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     3601 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     3661 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     3721 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     3781 ggacgtggtt ttcctttgaa aaacacgatg ataagcttgc cacaaccctt accggtcgcc
     3841 accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     3901 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     3961 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     4021 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     4081 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     4141 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     4201 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     4261 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     4321 atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga
     4381 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     4441 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     4501 agatactgcg acctccctag caaactgggg cacaagctta attgattcta gagtcgaccg
     4561 agcatcttac cgccatttat acccatattt gttctgtttt tcttgatttg ggtatacatt
     4621 taaatgttaa tagaacaaaa tggtggggca atcatttaca tttttaggga tatgtaatta
     4681 ctagttcagg tgtattgcca caagacaaac atgttaagaa actttcccgt tatttacgct
     4741 ctgttcctgt taatcaacct ctggattaca aaatttgtga aagattgact gatattctta
     4801 actatgttgc tccttttacg ctgtgtggat atgctgcttt atagcctctg tatctagcta
     4861 ttgcttcccg tacggctttc gttttctcct ccttgtataa atcctggttg ctgtctcttt
     4921 tagaggagtt gtggcccgtt gtccgtcaac gtggcgtggt gtgctctgtg tttgctgacg
     4981 caacccccac tggctggggc attgccacca cctgtcaact cctttctggg actttcgctt
     5041 tccccctccc gatcgccacg gcagaactca tcgccgcctg ccttgcccgc tgctggacag
     5101 gggctaggtt gctgggcact gataattccg tggtgttgtc atcggtacct ttttaaaaga
     5161 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatca taacttcgta
     5221 tagcatacat tatacgaagt tataatttat ttgtgaaatt tgtgatgcta ttgctttatt
     5281 tgtaaccata tgtttatttg tgaaatttgt gatgctattg ctttatttgt aaccattgct
     5341 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     5401 actagggaac ccactgctta agcctcaata aagcttgcct cgaccagcct cgactgtgcc
     5461 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5521 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5581 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5641 caatagcagg catgctgggg atgcggtggg ctctatggcc tgcagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5761 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5821 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6361 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6481 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6601 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6661 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6721 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6781 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6841 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6901 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6961 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7021 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7081 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7141 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7201 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7261 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7321 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7381 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7441 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7501 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7561 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7621 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc t
//
