LOCUS       sequence-271117-        6413 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 6413)
  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 6413)
  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..6413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(309..328)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(310..328)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2751..3323
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2774..3325
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2918..2935)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             3325..4029
                     /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    4108..4229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4119..4138)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4173..4192
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4331..4349
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4659..4676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4830..5418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4910..4929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5561..6370)
                     /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     6284..6303
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6362..6381
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gttcatgatg cagctggatc cagatcccta tacagttgaa gtcggaagtt tacatacact
       61 taagttggag tcattaaaac tcgtttttca actactccac aaatttcttg ttaacaaaca
      121 atagttttgg caagtcagtt aggacatcta ctttgtgcat gacacaagtc atttttccaa
      181 caattgttta cagacagatt atttcactta taattcactg tatcacaatt ccagtgggtc
      241 agaagtttac atacactaag ttgactgtgc ctttaaacag cttggaaaat tccagaaaat
      301 gatggtctcc ctatagtgag tcgtattaga tcccgggatt actagtttaa acctaggatg
      361 cataccggtc aagctagcga taattcctgc aggagtcaat gggaaaaacc cattggagcc
      421 aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag
      481 ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc
      541 caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt
      601 actgacatgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt
      661 caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga
      721 ctttccattg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc
      781 ccattattgg cacatacata aggtcaatag gggtgactag tggagaagag catgcttgag
      841 ggctgagtgc ccctcagtgg gcagagagca catggcccac agtccctgag aagttggggg
      901 gaggggtggg caattgaact ggtgcctaga gaaggtgggg cttgggtaaa ctgggaaagt
      961 gatgtggtgt actggctcca cctttttccc cagggtgggg gagaaccata tataagtgca
     1021 gtagtctctg tgaacattca agcttctgcc ttctccctcc tgtgagtttg gtaagtcact
     1081 gactgtctat gactgggaaa gggtgggcag gagatggggc agtgcaggaa aagtggcact
     1141 atgaaccctg cagccctagg aatgcatcta gcgataagct tgcatgcctg caggtcgagt
     1201 gtacaatggt agatggagtg atgattcttc ctgtgcttat catgattgct ctcccctccc
     1261 ctagtatgga agatgagaag cccaaggtca accccaaact ctacatgtgt gtgtgtgaag
     1321 gtctctcctg cggtaatgag gaccactgtg aaggccagca gtgcttttcc tcactgagca
     1381 tcaacgatgg cttccacgtc taccagaaag gctgcttcca ggtttatgag cagggaaaga
     1441 tgacctgtaa gaccccgccg tcccctggcc aagctgtgga gtgctgccaa ggggactggt
     1501 gtaacaggaa catcacggcc cagctgccca ctaaaggaaa atccttccct ggaacacaga
     1561 atttccactt ggaggttggc ctcattattc tctctgtagt gttcgcagta tgtcttttag
     1621 cctgcctgct gggagttgct ctccgaaaat ttaaaaggcg caaccaagaa cgcctcaatc
     1681 cccgagacgt ggagtatggc actatcgaag ggctcatcac caccaatgtt ggagacagca
     1741 ctttagcaga tttattggat cattcgtgta catcaggaag tggctctggt cttccttttc
     1801 tggtacaaag aacagtggct cgccagatta cactgttgga gtgtgtcggg aaaggcaggt
     1861 atggtgaggt gtggaggggc agctggcaag gggaaaatgt tgccgtgaag atcttctcct
     1921 cccgtgatga gaagtcatgg ttcagggaaa cggaattgta caacactgtg atgctgaggc
     1981 atgaaaatat cttaggtttc attgcttcag acatgacatc aagacactcc agtacccagc
     2041 tgtggttaat tacacattat catgaaatgg gatcgttgta cgactatctt cagcttacta
     2101 ctctggatac agttagctgc cttcgaatag tgctgtccat agctagtggt cttgcacatt
     2161 tgcacataga gatatttggg acccaagtga aaccagccat tgcccatcga gatttaaaga
     2221 gcaaaaatat tctggttaag aagaatggac agtgttgcat agcagatttg ggcctggcag
     2281 tcatgcattc ccagagcacc aatcagcttg atgtggggaa caatccccgt gtgggcacca
     2341 agcgctacat ggcccccgaa gttctagatg aaaccatcca ggtggattgt ttcgattctt
     2401 ataaaagggt cgatatttgg gcctttggac ttgttttgtg ggaagtggcc aggcggatgg
     2461 tgagcaatgg tatagtggag gattacaagc caccgttcta cgatgtggtt cccaatgacc
     2521 caagttttga agatatgagg aaggtagtct gtgtggatca acaaaggcca aacataccca
     2581 acagatggtt ctcagacccg acattaacct ctctggccaa gctaatgaaa gaatgctggt
     2641 atcaaaatcc atccgcaaga ctcacagcac tgcgtatcaa aaagactttg accaaaattg
     2701 ataattccct cgacaaattg aaaactgact gttgaggatc ccggtcgagg cccctctccc
     2761 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     2821 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     2881 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     2941 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     3001 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     3061 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     3121 tggatagttg tggaaagagt caaatggctc acctcaagcg tattcaacaa ggggctgaag
     3181 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     3241 acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     3301 tcctttgaaa aacacgatga taatatggtg ggtgaggata gcgtgctgat caccgagaac
     3361 atgcacatga aactgtacat ggagggcacc gtgaacgacc accacttcaa gtgcacatcc
     3421 gagggcgaag gcaagcccta cgagggcacc cagaccatga agatcaaggt ggtcgagggc
     3481 ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg cagcaaaacc
     3541 tttatcaacc acacccaggg catccccgac ttctttaagc agtccttccc tgagggcttc
     3601 acatgggaga ggatcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     3661 agcctccaga acggctgcct catctacaac gtcaagatca acggggtgaa cttcccatcc
     3721 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccagcaccga gatgctgtac
     3781 cccgctgaca gcggcctgag aggccatagc cagatggccc tgaagctcgt gggcgggggc
     3841 tacctgcact gctccctcaa gaccacatac agatccaaga aacccgctaa gaacctcaag
     3901 atgcccggct tctacttcgt ggacaggaga ctggaaagaa tcaaggaggc cgacaaagag
     3961 acctacgtcg agcagcacga gatggctgtg gccaggtact gcgacctgcc tagcaaactg
     4021 gggcacagct ccggactcag ataacgtacg tgcgcgcgaa tgcatccaga tctaggtacc
     4081 agggcccagc tagctggcca gacatgataa gatacattga tgagtttgga caaaccacaa
     4141 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4201 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4261 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     4321 tggaattatc aattaaccct cactaaaggg agaccaagtt aaacaattta aaggcaatgc
     4381 taccaaatac taattgagtg tatgtaaact tctgacccac tgggaatgtg atgaaagaaa
     4441 taaaagctga aatgaatcat tctctctact attattctga tatttcacat tcttaaaata
     4501 aagtggtgat cctaactgac ctaagacagg gaatttttac taggattaaa tgtcaggaat
     4561 tgtgaaaaag tgagtttaaa tgtatttggc taaggtgtat gtaaacttcc gacttcaact
     4621 gtatagggat ctggtaccat ttaaatctgt tccgcttcct cgctcactga ctcgctgcgc
     4681 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4741 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4801 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4861 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4921 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4981 tacctgtccg cctttctccc ttcgggaacc gtggcgcttt ctcatagctc acgctgtagg
     5041 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5101 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5161 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5221 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5281 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5341 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5401 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5461 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5521 atcctttttg ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca
     5581 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     5641 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     5701 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     5761 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     5821 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     5881 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     5941 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     6001 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     6061 ctgtttttcc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     6121 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     6181 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     6241 tcccatacaa gcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     6301 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgacgtt tcccgttgaa
     6361 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt att
//
