LOCUS       sequence-338550-        6822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCherry-TOSI (TOr-Signal-Indicator) is a fluorescent reporter for 
            mTORC1 signaling (monitoring PDCD4 degradation) which can be 
            introduced to mammalian cells by retrovirus vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6822)
  AUTHORS   Oki T, Mercier F, Kato H, Jung Y, McDonald TO, Spencer JA, Mazzola 
            MC, van Gastel N, Lin CP, Michor F, Kitamura T, Scadden DT
  TITLE     Imaging dynamic mTORC1 pathway activity in vivo reveals marked 
            shifts that support time-specific inhibitor therapy in AML.
  JOURNAL   Nat Commun. 2021 Jan 11;12(1):245. doi: 10.1038/s41467-020-20491-8.
  PUBMED    33431855
REFERENCE   2  (bases 1 to 6822)
  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: "Nat 
            Commun."; date: "2021-01-11"; volume: "12(1):245. doi"; pages: " 
            10.1038/s41467-020-20491-8"
COMMENT     Sequence Label: pMXs-IP-mCherry-TOSI
FEATURES             Location/Qualifiers
     source          1..6822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(9..26)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     236..255
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             429..1031
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MVTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     928..948
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1202..1671
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1801..1818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1972..2560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2052..2071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2724..3515,3516..3584))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3347..3366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3585..3689)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             3719..4312
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    4375..4732
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4791..5207
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     5137..5159
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     5175..5191
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    5217..5591
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     5529..5545
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      5620..5629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5626..6333
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5771..5789)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6039..6058
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6364..6384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    join(6664..6822,1..416,417..419,420..428)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
ORIGIN
        1 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
       61 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
      121 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
      181 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
      241 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
      301 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
      361 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
      421 ccacaaccat ggttaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
      481 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
      541 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
      601 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
      661 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
      721 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
      781 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
      841 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
      901 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
      961 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     1021 ccggtgcctg agtcgacgat aaaataaaag attttattta gtctccagaa aaagggggga
     1081 atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca
     1141 tggaaaaata cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca
     1201 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     1261 agaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     1321 cccggctcag ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta
     1381 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     1441 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     1501 taaaagagcc cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg
     1561 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     1621 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc acatgcagca
     1681 tgtatcaaaa ttaatttggt tttttttctt aagtatttac attaaatggc catagttgca
     1741 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1801 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1861 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1921 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1981 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2041 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2101 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2161 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2221 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2281 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2341 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2401 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2461 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2521 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2581 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2641 atcaaaaagg atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata
     2701 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2761 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2821 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2881 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2941 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3001 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3061 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3121 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3181 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3241 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3301 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3361 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3421 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3481 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3541 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3661 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgcataa
     3721 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
     3781 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca gatggaacag
     3841 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     3901 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     3961 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
     4021 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     4081 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     4141 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gattgactga gtcgcccggg
     4201 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     4261 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
     4321 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
     4381 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
     4441 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
     4501 gttcggaaca cccggccgca accctgggag acgtcccagg gacttcgggg gccgtttttg
     4561 tggcccgacc tgagtccaaa aatcccgatc gttttggact ctttggtgca cccccctaat
     4621 aggagggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
     4681 aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg
     4741 ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc tgaaaattag ggccagactg
     4801 ttaccactcc cttaagtttg accttaggtc actggaaaga tgtcgagcgg atcgctcaca
     4861 accagtcggt agatgtcaag aagagacgtt gggttacctt ctgctctgca gaatggccaa
     4921 cctttaacgt cggatggccg cgagacggca cctttaaccg agacctcatc acccaggtta
     4981 agatcaaggt cttttcacct ggcccgcatg gacacccaga ccaggtcccc tacatcgtga
     5041 cctgggaagc cttggctttt gacccccctc cctgggtcaa gccctttgta caccctaagc
     5101 ctccgcctcc tcttcctcca tccgccccgt ctctccccct tgaacctcct cgttcgaccc
     5161 cgcctcgatc ctccctttat ccagccctca ctccttctct aggcgccccc atatggccat
     5221 atgagatctt atatggggca cccccgcccc ttgtaaactt ccctgaccct gacatgacaa
     5281 gagttactaa cagcccctct ctccaagctc acttacaggc tctctactta gtccagcacg
     5341 aagtctggag acctctggcg gcagcctacc aagaacaact ggaccgaccg gtggtacctc
     5401 acccttaccg agtcggcgac acagtgtggg tccgccgaca ccagactaag aacctagaac
     5461 ctcgctggaa aggaccttac acagtcctgc tgaccacccc caccgccctc aaagtagacg
     5521 gcatcgcagc ttggatacac gccgcccacg tgaaggctgc cgaccccggg ggtggaccat
     5581 cctctagact gccggatcta gctagttaat taaggatccg ccaccatggt gagcaagggc
     5641 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     5701 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     5761 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     5821 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     5881 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     5941 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     6001 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     6061 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     6121 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     6181 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     6241 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     6301 cactccaccg gcggcatgga cgagctgtac aagggatatc catcacactg gcggccggaa
     6361 ttgccaaaaa agaagagaaa ggtagaattc atggatatag aaaatgagca gacactgaat
     6421 gtgaacccca ctgaccctga caatttaagc gactctctct tttctggaga tgaagaaaat
     6481 gctggcactg aagaaataaa gaatgaaata aatggaaatt ggatttccgc atctactatt
     6541 aatgaagcta gaatcaatgc aaaagccaaa agacgactgc ggaaaaattc atcccgggac
     6601 tctggccgag gagactcagt cagtgacaat tgagcggccg ctacgtaaat tccgcccccc
     6661 ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     6721 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     6781 ccggaaacct ggccctgtct tcttgacgag cattcctagg gg
//
