LOCUS       sequence-259378-        6424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ACVR1 with a G328V 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 6424)
  AUTHORS   Mendez F, Kadiyala P, Nunez FJ, Carney S, Nunez FM, Gauss JC, 
            Ravindran R, Pawar S, Edwards M, Garcia-Fabiani MB, Haase S, 
            Lowenstein PR, Castro MG
  TITLE     Therapeutic Efficacy of Immune Stimulatory Thymidine Kinase and 
            fms-like Tyrosine Kinase 3 Ligand (TK/Flt3L) Gene Therapy in a Mouse
            Model of High-Grade Brainstem Glioma.
  JOURNAL   Clin Cancer Res. 2020 Aug 1;26(15):4080-4092. doi: 
            10.1158/1078-0432.CCR-19-3714. Epub 2020 Apr 24.
  PUBMED    32332014
REFERENCE   2  (bases 1 to 6424)
  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"; doi: 
            "10.1158/1078-0432.CCR-19-3714"; journalName: "Clin Cancer Res"; 
            date: "2020-08-1- 1"; volume: "26"; issue: "15"; pages: "4080-4092"
COMMENT     Sequence Label: pKT2-ACVR1-G328V-IRES-Katushka
FEATURES             Location/Qualifiers
     source          1..6424
                     /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    2749..3321
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2772..3323
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2916..2933)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             3323..4027
                     /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    4106..4227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4117..4136)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4171..4190
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4329..4347
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4657..4674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4828..5416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4908..4927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5559..6368)
                     /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     6282..6301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6360..6379
                     /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 gcatctagaa tcgataagct tgcatgcctg caggtcgagt gtacaatggt
     1201 agatggagtg atgattcttc ctgtgcttat catgattgct ctcccctccc ctagtatgga
     1261 agatgagaag cccaaggtca accccaaact ctacatgtgt gtgtgtgaag gtctctcctg
     1321 cggtaatgag gaccactgtg aaggccagca gtgcttttcc tcactgagca tcaacgatgg
     1381 cttccacgtc taccagaaag gctgcttcca ggtttatgag cagggaaaga tgacctgtaa
     1441 gaccccgccg tcccctggcc aagccgtgga gtgctgccaa ggggactggt gtaacaggaa
     1501 catcacggcc cagctgccca ctaaaggaaa atccttccct ggaacacaga atttccactt
     1561 ggaggttggc ctcattattc tctctgtagt gttcgcagta tgtcttttag cctgcctgct
     1621 gggagttgct ctccgaaaat ttaaaaggcg caaccaagaa cgcctcaatc cccgagacgt
     1681 ggagtatggc actatcgaag ggctcatcac caccaatgtt ggagacagca ctttagcaga
     1741 tttattggat cattcgtgta catcaggaag tggctctggt cttccttttc tggtacaaag
     1801 aacagtggct cgccagatta cactgttgga gtgtgtcggg aaaggcaggt atggtgaggt
     1861 gtggaggggc agctggcaag gggagaatgt tgccgtgaag atcttctcct cccgtgatga
     1921 gaagtcatgg ttcagggaaa cggaattgta caacactgtg atgctgaggc atgaaaatat
     1981 cttaggtttc attgcttcag acatgacatc aagacactcc agtacccagc tgtggttaat
     2041 tacacattat catgaaatgg gatcgttgta cgactatctt cagcttacta ctctggatac
     2101 agttagctgc cttcgaatag tgctgtccat agctagtggt cttgcacatt tgcacataga
     2161 gatatttggg acccaagtga aaccagccat tgcccatcga gatttaaaga gcaaaaatat
     2221 tctggttaag aagaatggac agtgttgcat agcagatttg ggcctggcag tcatgcattc
     2281 ccagagcacc aatcagcttg atgtggggaa caatccccgt gtgggcacca agcgctacat
     2341 ggcccccgaa gttctagatg aaaccatcca ggtggattgt ttcgattctt ataaaagggt
     2401 cgatatttgg gcctttggac ttgttttgtg ggaagtggcc aggcggatgg tgagcaatgg
     2461 tatagtggag gattacaagc caccgttcta cgatgtggtt cccaatgacc caagttttga
     2521 agatatgagg aaggtagtct gtgtggatca acaaaggcca aacataccca acagatggtt
     2581 ctcagacccg acattaacct ctctggccaa gctaatgaaa gaatgctggt atcaaaatcc
     2641 atccgcaaga ctcacagcac tgcgtatcaa aaagactttg accaaaattg ataattccct
     2701 cgacaaattg aaaactgact gttgatctag aggatcccgg gctcgaggcc cctctccctc
     2761 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     2821 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     2881 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     2941 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     3001 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     3061 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     3121 gatagttgtg gaaagagtca aatggctcac ctcaagcgta ttcaacaagg ggctgaagga
     3181 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     3241 atgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     3301 ctttgaaaaa cacgatgata atatggtggg tgaggatagc gtgctgatca ccgagaacat
     3361 gcacatgaaa ctgtacatgg agggcaccgt gaacgaccac cacttcaagt gcacatccga
     3421 gggcgaaggc aagccctacg agggcaccca gaccatgaag atcaaggtgg tcgagggcgg
     3481 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcaaaacctt
     3541 tatcaaccac acccagggca tccccgactt ctttaagcag tccttccctg agggcttcac
     3601 atgggagagg atcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     3661 cctccagaac ggctgcctca tctacaacgt caagatcaac ggggtgaact tcccatccaa
     3721 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc agcaccgaga tgctgtaccc
     3781 cgctgacagc ggcctgagag gccatagcca gatggccctg aagctcgtgg gcgggggcta
     3841 cctgcactgc tccctcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
     3901 gcccggcttc tacttcgtgg acaggagact ggaaagaatc aaggaggccg acaaagagac
     3961 ctacgtcgag cagcacgaga tggctgtggc caggtactgc gacctgccta gcaaactggg
     4021 gcacagctcc ggactcagat aacgtacgtg cgcgcgaatg catccagatc taggtaccag
     4081 ggcccagcta gctggccaga catgataaga tacattgatg agtttggaca aaccacaact
     4141 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     4201 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     4261 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     4321 gaattatcaa ttaaccctca ctaaagggag accaagttaa acaatttaaa ggcaatgcta
     4381 ccaaatacta attgagtgta tgtaaacttc tgacccactg ggaatgtgat gaaagaaata
     4441 aaagctgaaa tgaatcattc tctctactat tattctgata tttcacattc ttaaaataaa
     4501 gtggtgatcc taactgacct aagacaggga atttttacta ggattaaatg tcaggaattg
     4561 tgaaaaagtg agtttaaatg tatttggcta aggtgtatgt aaacttccga cttcaactgt
     4621 atagggatct ggtaccattt aaatctgttc cgcttcctcg ctcactgact cgctgcgctc
     4681 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4741 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4801 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4861 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4921 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4981 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5041 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5101 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5161 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5221 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5281 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5341 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5401 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5461 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5521 cctttttgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc
     5581 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     5641 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     5701 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     5761 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     5821 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     5881 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     5941 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     6001 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     6061 gtttttccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     6121 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     6181 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     6241 ccatacaagc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     6301 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgacgtttc ccgttgaata
     6361 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     6421 gcag
//
