LOCUS       sequence-248702-        7371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA targeting ATRX with a GFP reporter which is driven 
            by the SV40 promoter. Construct has inverted repeats to be used in 
            Sleeping beauty system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7371)
  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 7371)
  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: pT2-shATRX-GFP
FEATURES             Location/Qualifiers
     source          1..7371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..107
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     99..116
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     100..116
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     126..145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        126..144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             531..1047
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1111..1452
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1519..1935
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1865..1887
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1903..1919
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1975..2096
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2124..2138
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2166..2292
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(2325..2345)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2330..2659
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2510..2645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(2666..2668,2669..2671,2672..3385)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2711..2732)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2972..2991)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3319..3340
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3458..3972
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4134..4150
                     /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(4158..4164,4165..4182,4183..4188))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4203..4224
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4683..4703)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4683..4701)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4722..4738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4722..4738)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4746..4762
                     /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(4770..4776,4777..4794,4795..4800))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4815..4836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4953..4970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5124..5712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5204..5223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5883..6674,6675..6743))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6506..6525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6744..6848)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6875..7330
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6962..6981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7172..7193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
       61 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
      121 gcgcgtaata cgactcacta tagggcgaat tggagctcgg atccctatac agttgaagtc
      181 ggaagtttac atacacttaa gttggagtca ttaaaactcg tttttcaact actccacaaa
      241 tttcttgtta acaaacaata gttttggcaa gtcagttagg acatctactt tgtgcatgac
      301 acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa ttcactgtat
      361 cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt taaacagctt
      421 ggaaaattcc agaaaatgat gtcatggctt tagaagcttg atctgaagcc tatagagtac
      481 gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
      541 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata
      601 cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg
      661 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
      721 tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
      781 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
      841 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
      901 gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa taaagcctct
      961 tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct cagattgatt
     1021 gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga gacccctgcc
     1081 cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt cgtgtctgtc
     1141 tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct gtactagtta
     1201 gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc
     1261 cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga
     1321 agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac
     1381 ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg
     1441 tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg
     1501 tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac
     1561 tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg
     1621 gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc
     1681 tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga
     1741 cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc
     1801 tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct
     1861 ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact
     1921 ccttctctag gcgccggaat tagatctctc gactagggat aacagggtaa ttgtttgaat
     1981 gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta
     2041 cttcttcagg ttaacccaac agaaggctcg agaaggtata ttgctgttga cagtgagcgc
     2101 ccgcttggat ggttctacta atagtgaagc cacagatgta ttagtagaac catccaagcg
     2161 gttgcctact gcctcggaat tcaaggggct actttaggag caattatctt gtttactaaa
     2221 actgaatacc ttgctatctc tttgatacat ttttacaaag ctgaattaaa atggtataaa
     2281 ttaaatcact tttttcaatt cgccagcaca gtggtcgacc ctgtggaatg tgtgtcagtt
     2341 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2401 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2461 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     2521 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2581 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     2641 aggcctaggc ttttgcaaaa agctcatggt gagcaagggc gaggagctgt tcaccggggt
     2701 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtctgg
     2761 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2821 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2881 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2941 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3001 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3061 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3121 tatcatggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat
     3181 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3241 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     3301 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3361 cggcatggac gagctgtaca agtaatgaat taattaagaa ttatcaagct tatcgataaa
     3421 ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt
     3481 tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata
     3541 gagaagttca gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat
     3601 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt
     3661 cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga
     3721 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg
     3781 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc
     3841 cgatagactg cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg
     3901 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg
     3961 ggggtctttc atgggtaaca gtttcttgaa gttggagaac aacattctga gggtaggagt
     4021 cgaatattaa gtaatcctga ctcaattagc cactgttttg aatccacata ctccaatact
     4081 cctgaaatag ttcattatgg acagcgcaga aagagctggg gagaattgtg aaattgttat
     4141 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4201 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4261 aacctgtcgt gccagatcca tggacttcac tagtgcgcgc ggccgctcta gatggccaga
     4321 tctagcttgt ggaaggctac tcgaaatgtt tgacccaagt taaacaattt aaaggcaatg
     4381 ctaccaaata ctaattgagt gtatgtaaac ttctgaccca ctgggaatgt gatgaaagaa
     4441 ataaaagctg aaatgaatca ttctctctac tattattctg atatttcaca ttcttaaaat
     4501 aaagtggtga tcctaactga cctaagacag ggaattttta ctaggattaa atgtcaggaa
     4561 ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta tgtaaacttc cgacttcaac
     4621 tgtataggga tcctctagct agagtcgacc tcgcgggggg gcccggtacc cagcttttgt
     4681 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     4741 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4801 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4861 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4921 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4981 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5041 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5101 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5161 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5221 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5281 tccgcctttc tcccttcggg aaccgtggcg ctttctcata gctcacgctg taggtatctc
     5341 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5461 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5521 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5581 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5641 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5701 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5761 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5821 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5881 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5941 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6001 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6061 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6181 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta acttggccgc agtgttatca
     6361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6481 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6841 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
     6901 gcggcgggag tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6961 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     7021 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     7081 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     7141 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     7201 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     7261 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     7321 cttacaattt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga t
//
