LOCUS       sequence-66678-e        6801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses I-Sce I nuclease and BFP tracker in mammalian cells, codes
            for eGFP donor sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6801)
  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 6801)
  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: pEX.d14GFP.sEF1a.Kozak.HA.NLS.Sce.T2A.BFP
FEATURES             Location/Qualifiers
     source          1..6801
                     /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"
     CDS             join(234..236,237..239,240..752)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(279..300)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(540..559)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     887..908
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        938..1149
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1158..1167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1173..1199
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1173..1199
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1209..1229
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1950..2003
                     /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             2004..2705
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    2719..3307
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2772..2792)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3190..3201)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3381..3398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3387..3611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3657..4085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3744..3763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3954..3975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4094..4114)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     4173..4192
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4477..4598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4514..4533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4568..4587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4647..4663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4647..4663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4660..4682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4671..4687
                     /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(4695..4701,4702..4719,4720..4725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4740..4761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4878..4895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5049..5634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5129..5148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5805..6596,6597..6665))
                     /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     6428..6447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6666..6770)
                     /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 cgtatggtga
      241 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      301 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
      361 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
      421 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
      481 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
      541 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
      601 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
      661 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
      721 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
      781 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      841 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      901 agttcgtgtg aggatccagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      961 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     1021 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     1081 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1141 agaacacagg atctgccgcc accatgggcg tataccccta cgacgtgccc gactacgccc
     1201 ccgggccccc taagaaaaag aggaaggtga agaacatcaa gaagaaccag gtcatgaacc
     1261 tgggccccaa cagcaagctg ctgaaggagt acaagagcca gctgatcgag ctgaacatcg
     1321 agcagttcga ggccggcatc ggcctgatcc tgggcgacgc ctacatcagg agcagggacg
     1381 agggcaagac ctactgcatg cagttcgagt ggaagaacaa ggcctacatg gaccacgtgt
     1441 gcctgctgta cgaccagtgg gtgctgagcc ccccccacaa gaaggagagg gtgaaccacc
     1501 tgggcaacct ggtcatcacc tggggcgccc agaccttcaa gcaccaggcc ttcaacaagc
     1561 tggccaacct gttcatcgtg aacaacaaga agaccatccc caacaacctg gtggagaact
     1621 acctgacccc catgagcctg gcctactggt tcatggacga cggcggcaag tgggactaca
     1681 acaagaacag caccaacaag agcatcgtgc tgaacaccca gagcttcacc ttcgaggagg
     1741 tggagtacct ggtgaagggc ctgaggaaca agttccagct gaactgctac gtgaagatca
     1801 acaagaacaa gcccatcatc tacatcgaca gcatgagcta cctgatcttc tacaacctga
     1861 tcaagcccta cctgatcccc cagatgatgt acaagctgcc caacaccatc agcagcgaga
     1921 ccttcctgaa gggcggcggc ggatccggtg agggcagagg aagtcttcta acatgcggtg
     1981 acgtggagga gaatccgggc cccatgagcg agctgattaa ggagaacatg cacatgaagc
     2041 tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca
     2101 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     2161 tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca
     2221 cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag
     2281 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     2341 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga
     2401 tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg
     2461 gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa
     2521 acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct
     2581 actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc
     2641 agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta
     2701 attgattcta gagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2761 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2821 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2881 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2941 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3001 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3061 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     3121 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3181 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3241 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3301 tccccgcctg gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     3361 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3421 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3481 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3541 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3601 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3721 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3841 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3961 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4021 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4081 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4141 ggcagaagta tgcaaagcct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4201 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4261 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4321 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4381 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4441 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4501 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4561 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4621 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4681 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4741 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4801 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4861 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4921 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4981 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5041 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5101 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5161 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5221 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5281 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5341 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5401 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5461 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5521 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5581 agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5641 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5701 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5761 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5821 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5881 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5941 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6001 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6061 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6121 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6181 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6241 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6301 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6361 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6421 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6481 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6541 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6601 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6661 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6721 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6781 cgaaaagtgc cacctgacgt c
//
