LOCUS       sequence-67129-e        6112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses I-Sce I nuclease in mammalian cells and BFP as a tracker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6112)
  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 6112)
  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.sEF1a.Kozak.HA.NLS.Sce.T2A.BFP
FEATURES             Location/Qualifiers
     source          1..6112
                     /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"
     promoter        249..460
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      469..478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             484..510
                     /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             484..510
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             520..540
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1261..1314
                     /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             1315..2016
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    2030..2618
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2083..2103)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2501..2512)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2692..2709)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2698..2922
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2968..3396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3055..3074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3265..3286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3405..3425)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     3484..3503
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3788..3909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3825..3844)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3879..3898
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3958..3974)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3958..3974)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3971..3993)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3982..3998
                     /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(4006..4012,4013..4030,4031..4036))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4051..4072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4189..4206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4360..4945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4440..4459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5116..5907,5908..5976))
                     /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     5739..5758
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5977..6081)
                     /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 cagatataag gctccggtgc
      241 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      301 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      361 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      421 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gatctgccgc caccatgggc
      481 gtatacccct acgacgtgcc cgactacgcc cccgggcccc ctaagaaaaa gaggaaggtg
      541 aagaacatca agaagaacca ggtcatgaac ctgggcccca acagcaagct gctgaaggag
      601 tacaagagcc agctgatcga gctgaacatc gagcagttcg aggccggcat cggcctgatc
      661 ctgggcgacg cctacatcag gagcagggac gagggcaaga cctactgcat gcagttcgag
      721 tggaagaaca aggcctacat ggaccacgtg tgcctgctgt acgaccagtg ggtgctgagc
      781 cccccccaca agaaggagag ggtgaaccac ctgggcaacc tggtcatcac ctggggcgcc
      841 cagaccttca agcaccaggc cttcaacaag ctggccaacc tgttcatcgt gaacaacaag
      901 aagaccatcc ccaacaacct ggtggagaac tacctgaccc ccatgagcct ggcctactgg
      961 ttcatggacg acggcggcaa gtgggactac aacaagaaca gcaccaacaa gagcatcgtg
     1021 ctgaacaccc agagcttcac cttcgaggag gtggagtacc tggtgaaggg cctgaggaac
     1081 aagttccagc tgaactgcta cgtgaagatc aacaagaaca agcccatcat ctacatcgac
     1141 agcatgagct acctgatctt ctacaacctg atcaagccct acctgatccc ccagatgatg
     1201 tacaagctgc ccaacaccat cagcagcgag accttcctga agggcggcgg cggatccggt
     1261 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatccggg ccccatgagc
     1321 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     1381 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     1441 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     1501 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     1561 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     1621 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     1681 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1741 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     1801 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     1861 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     1921 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     1981 gacctcccta gcaaactggg gcacaagctt aattgattct agagtcgaca atcaacctct
     2041 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2101 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2161 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2221 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2281 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2341 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2401 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     2461 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2521 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     2581 tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattctgc agatatccag
     2641 cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2701 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2761 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2821 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2881 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2941 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3001 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3061 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3121 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3181 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3241 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3301 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3361 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3421 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagcc tatcaggaca
     3481 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3541 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3601 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     3661 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     3721 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     3781 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3841 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3901 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     3961 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4021 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4081 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4141 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4201 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4261 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4321 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4381 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4441 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4501 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4561 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4621 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4681 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4741 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4801 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4861 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca agcagcagat
     4921 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4981 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5041 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5101 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5161 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5221 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5281 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5341 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5401 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5461 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5521 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5581 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5641 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5701 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5761 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5821 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5881 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5941 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6001 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6061 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
