LOCUS       sequence-252091-        6158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses IDH1 with a R132H mutation and 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 6158)
  AUTHORS   Nunez FJ, Mendez FM, Kadiyala P, Alghamri MS, Savelieff MG, 
            Garcia-Fabiani MB, Haase S, Koschmann C, Calinescu AA, Kamran N, 
            Saxena M, Patel R, Carney S, Guo MZ, Edwards M, Ljungman M, Qin T, 
            Sartor MA, Tagett R, Venneti S, Brosnan-Cashman J, Meeker A, 
            Gorbunova V, Zhao L, Kremer DM, Zhang L, Lyssiotis CA, Jones L, 
            Herting CJ, Ross JL, Hambardzumyan D, Hervey-Jumper S, Figueroa ME, 
            Lowenstein PR, Castro MG
  TITLE     IDH1-R132H acts as a tumor suppressor in glioma via epigenetic 
            up-regulation of the DNA damage response.
  JOURNAL   Sci Transl Med. 2019 Feb 13;11(479). pii: 11/479/eaaq1427. doi: 
            10.1126/scitranslmed.aaq1427.
  PUBMED    30760578
REFERENCE   2  (bases 1 to 6158)
  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: "Sci Transl 
            Med."; date: "2019-02-13"; volume: "11(479). pii: 11/479/eaaq1427. 
            doi"; pages: " 10.1126/scitranslmed.aaq1427"
COMMENT     Sequence Label: pKT-IDH1(R132H)-IRES-Katushka
FEATURES             Location/Qualifiers
     source          1..6158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(296..315)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(297..315)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2483..3055
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2506..3057
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2650..2667)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             3057..3761
                     /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    3840..3961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3851..3870)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3905..3924
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4063..4081
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4391..4408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4562..5150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4642..4661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5293..6102)
                     /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     6016..6035
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6094..6113
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctggatccag atccctatac agttgaagtc ggaagtttac atacacttaa gttggagtca
       61 ttaaaactcg tttttcaact actccacaaa tttcttgtta acaaacaata gttttggcaa
      121 gtcagttagg acatctactt tgtgcatgac acaagtcatt tttccaacaa ttgtttacag
      181 acagattatt tcacttataa ttcactgtat cacaattcca gtgggtcaga agtttacata
      241 cactaagttg actgtgcctt taaacagctt ggaaaattcc agaaaatgat ggtctcccta
      301 tagtgagtcg tattagatcc cgggattact agtttaaacc taggatgcat accggtcaag
      361 ctagcgataa ttcctgcagg agtcaatggg aaaaacccat tggagccaag tacactgact
      421 caatagggac tttccattgg gttttgccca gtacataagg tcaatagggg gtgagtcaac
      481 aggaaagtcc cattggagcc aagtacattg agtcaatagg gactttccaa tgggttttgc
      541 ccagtacata aggtcaatgg gaggtaagcc aatgggtttt tcccattact gacatgtata
      601 ctgagtcatt agggactttc caatgggttt tgcccagtac ataaggtcaa taggggtgaa
      661 tcaacaggaa agtcccattg gagccaagta cactgagtca atagggactt tccattgggt
      721 tttgcccagt acaaaaggtc aatagggggt gagtcaatgg gtttttccca ttattggcac
      781 atacataagg tcaatagggg tgactagtgg agaagagcat gcttgagggc tgagtgcccc
      841 tcagtgggca gagagcacat ggcccacagt ccctgagaag ttggggggag gggtgggcaa
      901 ttgaactggt gcctagagaa ggtggggctt gggtaaactg ggaaagtgat gtggtgtact
      961 ggctccacct ttttccccag ggtgggggag aaccatatat aagtgcagta gtctctgtga
     1021 acattcaagc ttctgccttc tccctcctgt gagtttggta agtcactgac tgtctatgcc
     1081 tgggaaaggg tgggcaggag atggggcagt gcaggaaaag tggcactatg aaccctgcag
     1141 ccctaggaat gcatctagac aattgtacta accttcttct ctttcctctc ctgacaggtt
     1201 ggtgtacagc ggcccgccat gtccaaaaaa atcagtggcg gttctgtggt agagatgcaa
     1261 ggagatgaaa tgacacgaat catttgggaa ttgattaaag agaaactcat ttttccctac
     1321 gtggaattgg atctacatag ctatgattta ggcatagaga atcgtgatgc caccaacgac
     1381 caagtcacca aggatgctgc agaagctata aagaagcata atgttggcgt caaatgtgcc
     1441 actatcactc ctgatgagaa gagggttgag gagttcaagt tgaaacaaat gtggaaatca
     1501 ccaaatggca ccatacgaaa tattctgggt ggcacggtct tcagagaagc cattatctgc
     1561 aaaaatatcc cccggcttgt gagtggatgg gtaaaaccta tcatcatagg tcatcatgct
     1621 tatggggatc aatacagagc aactgatttt gttgttcctg ggcctggaaa agtagagata
     1681 acctacacac caagtgacgg aacccaaaag gtgacatacc tggtacataa ctttgaagaa
     1741 ggtggtggtg ttgccatggg gatgtataat caagataagt caattgaaga ttttgcacac
     1801 agttccttcc aaatggctct gtctaagggt tggcctttgt atctgagcac caaaaacact
     1861 attctgaaga aatatgatgg gcgttttaaa gacatctttc aggagatata tgacaagcag
     1921 tacaagtccc agtttgaagc tcaaaagatc tggtatgagc ataggctcat cgacgacatg
     1981 gtggcccaag ctatgaaatc agagggaggc ttcatctggg cctgtaaaaa ctatgatggt
     2041 gacgtgcagt cggactctgt ggcccaaggg tatggctctc tcggcatgat gaccagcgtg
     2101 ctggtttgtc cagatggcaa gacagtagaa gcagagtctg cccacgggac tgtaacccgt
     2161 cactaccgca tgtaccagaa aggacaggag acgtccacca atcccattgc ttccattttt
     2221 gcctggacca gagggttagc ccacagagca aagcttgata acaataaaga gcttgccttc
     2281 tttgcaaatg ctttggaaga agtctctatt gagacaattg aggctggctt catgaccaag
     2341 gacttggctg cttgcattaa aggtttaccc aatgtgcaac gttctgacta cttgaataca
     2401 tttgagttca tggataaact tggagaaaac ttgaagatca aactagctca ggccaaactt
     2461 acgcgtacgc ggccgctcga ggcccctctc cctccccccc ccctaacgtt actggccgaa
     2521 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     2581 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     2641 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     2701 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     2761 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     2821 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     2881 tcacctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     2941 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg
     3001 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     3061 tgggtgagga tagcgtgctg atcaccgaga acatgcacat gaaactgtac atggagggca
     3121 ccgtgaacga ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     3181 cccagaccat gaagatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     3241 tggctaccag cttcatgtac ggcagcaaaa cctttatcaa ccacacccag ggcatccccg
     3301 acttctttaa gcagtccttc cctgagggct tcacatggga gaggatcacc acatacgaag
     3361 acgggggcgt gctgaccgct acccaggaca ccagcctcca gaacggctgc ctcatctaca
     3421 acgtcaagat caacggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac
     3481 tcggctggga ggccagcacc gagatgctgt accccgctga cagcggcctg agaggccata
     3541 gccagatggc cctgaagctc gtgggcgggg gctacctgca ctgctccctc aagaccacat
     3601 acagatccaa gaaacccgct aagaacctca agatgcccgg cttctacttc gtggacagga
     3661 gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac gagatggctg
     3721 tggccaggta ctgcgacctg cctagcaaac tggggcacag ctccggactc agataacgta
     3781 cgtgcgcgcg aatgcatcca gatctaggta ccagggccca gctagctggc cagacatgat
     3841 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3901 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3961 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4021 ttaaagcaag taaaacctct acaaatgtgg tatggaatta tcaattaacc ctcactaaag
     4081 ggagaccaag ttaaacaatt taaaggcaat gctaccaaat actaattgag tgtatgtaaa
     4141 cttctgaccc actgggaatg tgatgaaaga aataaaagct gaaatgaatc attctctcta
     4201 ctattattct gatatttcac attcttaaaa taaagtggtg atcctaactg acctaagaca
     4261 gggaattttt actaggatta aatgtcagga attgtgaaaa agtgagttta aatgtatttg
     4321 gctaaggtgt atgtaaactt ccgacttcaa ctgtataggg atctggtacc atttaaatct
     4381 gttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4441 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4741 ccgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4981 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5161 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5221 tcatgagatt atcaaaaagg atcttcacct agatcctttt tgccagtgtt acaaccaatt
     5281 aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     5341 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     5401 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     5461 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     5521 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     5581 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     5641 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     5701 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     5761 tgaatcagga tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag
     5821 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     5881 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     5941 atgtttcaga aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc
     6001 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     6061 atttaatcgc ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact
     6121 gtttatgtaa gcagacagtt ttattgttca tgatgcag
//
