LOCUS       sequence-261114-        6305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses IDH1 with a R132H mutation and has Myc and FLAG tags. 
            IDH1(R132H) also has a katushka fluorescent reporter. Construct has 
            inverted repeats to be used in Sleeping Beauty transposon system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6305)
  TITLE     pKT-mIDH1(R132H)-Myc-FLAG-IRES-Katushka
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6305)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKT-mIDH1(R132H)-Myc-FLAG-IRES-Kat
FEATURES             Location/Qualifiers
     source          1..6305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2123..2152
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2171..2194
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2214..2230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2214..2230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    2251..2823
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2274..2825
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2418..2435)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             2825..3529
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     polyA_signal    3608..3729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3619..3638)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3673..3692
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3831..3849
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4159..4176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4330..4918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4410..4429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5061..5870)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5784..5803
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5862..5881
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(6222..6241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6223..6241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagtcaatgg gaaaaaccca ttggagccaa gtacactgac tcaataggga ctttccattg
       61 ggttttgccc agtacataag gtcaataggg ggtgagtcaa caggaaagtc ccattggagc
      121 caagtacatt gagtcaatag ggactttcca atgggttttg cccagtacat aaggtcaatg
      181 ggaggtaagc caatgggttt ttcccattac tgacatgtat actgagtcat tagggacttt
      241 ccaatgggtt ttgcccagta cataaggtca ataggggtga atcaacagga aagtcccatt
      301 ggagccaagt acactgagtc aatagggact ttccattggg ttttgcccag tacaaaaggt
      361 caataggggg tgagtcaatg ggtttttccc attattggca catacataag gtcaataggg
      421 gtgactagtg gagaagagca tgcttgaggg ctgagtgccc ctcagtgggc agagagcaca
      481 tggcccacag tccctgagaa gttgggggga ggggtgggca attgaactgg tgcctagaga
      541 aggtggggct tgggtaaact gggaaagtga tgtggtgtac tggctccacc tttttcccca
      601 gggtggggga gaaccatata taagtgcagt agtctctgtg aacattcaag cttctgcctt
      661 ctccctcctg tgagtttggt aagtcactga ctgtctatgc ctgggaaagg gtgggcagga
      721 gatggggcag tgcaggaaaa gtggcactat gaaccctgca gccctaggaa tgcatctagc
      781 gctaccggac tcagatctcg agctcaagct tcgaattcgt cgactggatc cggtaccgag
      841 gagatctgcc gccgcgatcg ccatgtccaa aaaaatcagt ggcggttctg tggtagagat
      901 gcaaggagat gaaatgacac gaatcatttg ggaattgatt aaagagaaac tcatttttcc
      961 ctacgtggaa ttggatctac atagctatga tttaggcata gagaatcgtg atgccaccaa
     1021 cgaccaagtc accaaggatg ctgcagaagc tataaagaag cataatgttg gcgtcaaatg
     1081 tgccactatc actcctgatg agaagagggt tgaggagttc aagttgaaac aaatgtggaa
     1141 atcaccaaat ggcaccatac gaaatattct gggtggcacg gtcttcagag aagccattat
     1201 ctgcaaaaat atcccccggc ttgtgagtgg atgggtaaaa cctatcatca taggtcatca
     1261 tgcttatggg gatcaataca gagcaactga ttttgttgtt cctgggcctg gaaaagtaga
     1321 gataacctac acaccaagtg acggaaccca aaaggtgaca tacctggtac ataactttga
     1381 agaaggtggt ggtgttgcca tggggatgta taatcaagat aagtcaattg aagattttgc
     1441 acacagttcc ttccaaatgg ctctgtctaa gggttggcct ttgtatctga gcaccaaaaa
     1501 cactattctg aagaaatatg atgggcgttt taaagacatc tttcaggaga tatatgacaa
     1561 gcagtacaag tcccagtttg aagctcaaaa gatctggtat gagcataggc tcatcgacga
     1621 catggtggcc caagctatga aatcagaggg aggcttcatc tgggcctgta aaaactatga
     1681 tggtgacgtg cagtcggact ctgtggccca agggtatggc tctctcggca tgatgaccag
     1741 cgtgctggtt tgtccagatg gcaagacagt agaagcagag tctgcccacg ggactgtaac
     1801 ccgtcactac cgcatgtacc agaaaggaca ggagacgtcc accaatccca ttgcttccat
     1861 ttttgcctgg accagagggt tagcccacag agcaaagctt gataacaata aagagcttgc
     1921 cttctttgca aatgctttgg aagaagtctc tattgagaca attgaggctg gcttcatgac
     1981 caaggacttg gctgcttgca ttaaaggttt acccaatgtg caacgttctg actacttgaa
     2041 tacatttgag ttcatggata aacttggaga aaacttgaag atcaaactag ctcaggccaa
     2101 acttacgcgt acgcggccgc tcgagcagaa actcatctca gaagaggatc tggcagcaaa
     2161 tgatatcctg gattacaagg atgacgacga taaggtttaa acggccggcc gcggtcatag
     2221 ctgtttcctg aacagatccc ccgctcgagg cccctctccc tccccccccc ctaacgttac
     2281 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     2341 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2401 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     2461 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     2521 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     2581 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     2641 caaatggctc acctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2701 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     2761 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     2821 taatatggtg ggtgaggata gcgtgctgat caccgagaac atgcacatga aactgtacat
     2881 ggagggcacc gtgaacgacc accacttcaa gtgcacatcc gagggcgaag gcaagcccta
     2941 cgagggcacc cagaccatga agatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     3001 cgacatcctg gctaccagct tcatgtacgg cagcaaaacc tttatcaacc acacccaggg
     3061 catccccgac ttctttaagc agtccttccc tgagggcttc acatgggaga ggatcaccac
     3121 atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccaga acggctgcct
     3181 catctacaac gtcaagatca acggggtgaa cttcccatcc aacggccctg tgatgcagaa
     3241 gaaaacactc ggctgggagg ccagcaccga gatgctgtac cccgctgaca gcggcctgag
     3301 aggccatagc cagatggccc tgaagctcgt gggcgggggc tacctgcact gctccctcaa
     3361 gaccacatac agatccaaga aacccgctaa gaacctcaag atgcccggct tctacttcgt
     3421 ggacaggaga ctggaaagaa tcaaggaggc cgacaaagag acctacgtcg agcagcacga
     3481 gatggctgtg gccaggtact gcgacctgcc tagcaaactg gggcacagct ccggactcag
     3541 ataacgtacg tgcgcgcgaa tgcatccaga tctaggtacc agggcccagc tagctggcca
     3601 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     3661 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     3721 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     3781 gaggtttttt aaagcaagta aaacctctac aaatgtggta tggaattatc aattaaccct
     3841 cactaaaggg agaccaagtt aaacaattta aaggcaatgc taccaaatac taattgagtg
     3901 tatgtaaact tctgacccac tgggaatgtg atgaaagaaa taaaagctga aatgaatcat
     3961 tctctctact attattctga tatttcacat tcttaaaata aagtggtgat cctaactgac
     4021 ctaagacagg gaatttttac taggattaaa tgtcaggaat tgtgaaaaag tgagtttaaa
     4081 tgtatttggc taaggtgtat gtaaacttcc gacttcaact gtatagggat ctggtaccat
     4141 ttaaatctgt tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4201 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4261 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4321 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4381 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4441 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4501 ttcgggaacc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4561 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4621 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4681 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4741 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4801 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4861 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4921 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4981 gattttggtc atgagattat caaaaaggat cttcacctag atcctttttg ccagtgttac
     5041 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     5101 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     5161 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     5221 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     5281 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     5341 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     5401 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     5461 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     5521 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgtttttcc ggggatcgca
     5581 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     5641 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     5701 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa gcgatagatt
     5761 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     5821 atgttggaat ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat aacacccctt
     5881 gtattactgt ttatgtaagc agacagtttt attgttcatg atgcagctgg atccagatcc
     5941 ctatacagtt gaagtcggaa gtttacatac acttaagttg gagtcattaa aactcgtttt
     6001 tcaactactc cacaaatttc ttgttaacaa acaatagttt tggcaagtca gttaggacat
     6061 ctactttgtg catgacacaa gtcatttttc caacaattgt ttacagacag attatttcac
     6121 ttataattca ctgtatcaca attccagtgg gtcagaagtt tacatacact aagttgactg
     6181 tgcctttaaa cagcttggaa aattccagaa aatgatggtc tccctatagt gagtcgtatt
     6241 agatcccggg attactagtt taaacctagg atgcataccg gtcaagctag cgataattcc
     6301 tgcag
//
