LOCUS       sequence-109685-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for TORCAR biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7225)
  AUTHORS   Zhou X, Clister TL, Lowry PR, Seldin MM, Wong GW, Zhang J
  TITLE     Dynamic Visualization of mTORC1 Activity in Living Cells.
  JOURNAL   Cell Rep. 2015 Mar 11. pii: S2211-1247(15)00177-1. doi: 
            10.1016/j.celrep.2015.02.031.
  PUBMED    25772363
REFERENCE   2  (bases 1 to 7225)
  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: "Cell Rep. 
            2015 Mar 11. pii: S2211-1247(15)00177-1. doi: 
            10.1016/j.celrep.2015.02.031."
COMMENT     Sequence Label: pcDNA3-TORCAR(T/A)
FEATURES             Location/Qualifiers
     source          1..7225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(914..916,917..919,920..1630)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(959..980)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1220..1239)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1567..1588
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2000..2710
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     promoter        complement(2777..2795)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2778..2795)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2815..2832)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2821..3045
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3091..3519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3178..3197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3388..3409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3528..3548)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3533..3863
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3714..3849
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3776..3795
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3930..4724
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3984..4003)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4594..4613
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4898..5019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4935..4954)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4989..5008
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5068..5084)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5068..5084)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5081..5103)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5092..5108
                     /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(5116..5122,5123..5140,5141..5146))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5161..5182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5299..5316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5470..6058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5550..5569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6229..7020,7021..7089))
                     /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     6852..6871
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7090..7194)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      961 ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgtccggcg agggcgaggg
     1021 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1081 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttcg cccgctaccc
     1141 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1201 gcgtaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1261 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1321 catcctgggg cacaagctgg agtacaacgc catcagtgac aacgtctata tcaccgccga
     1381 caagcagaag aacggcatca aggcccactt caagatccgc cacaacatcg aggacggcag
     1441 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1501 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1561 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1621 gctgtacaag cgcatgcata tgtccggggg cagcagctgc agccagaccc caagccgggc
     1681 catccccgcc actcgccggg tggtgctcgg cgacggcgtg cagctcccgc ccggggacta
     1741 cagcacggcc cccggcggca cgctcttcag caccgccccg ggaggtacca ggatcatcta
     1801 tgaccggaaa ttcctgatgg agtgtcggaa ctcacctgtg accaaaacac ccccaaggga
     1861 tctgcccacc attccggggg tcaccagccc ttccagtgat gagcccccca tggaagccag
     1921 ccagagccac ctgcgcaata gcccagaaga taagcgggcg ggcggtgaag agtcacagtt
     1981 tgagatggac attgagctct ctaaaggtga agaattattc actggtgttg tcccaatttt
     2041 ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg aaggtgaagg
     2101 tgatgctacg tacggtaaat tgaccttaaa attactctgt actactggta aattgccagt
     2161 tccatggcca accttagtca ctactttagg ttatggtgtt caatgttttg ctagataccc
     2221 agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt atgttcaaga
     2281 aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag tcaagtttga
     2341 aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag aagatggtaa
     2401 cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca tcactgctga
     2461 caaacaaaag aatggtatca aagctaactt caaaattaga cacaacattg aagatggtgg
     2521 tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt
     2581 accagacaac cattacttat cctatcaatc tgccttattc aaagatccaa acgaaaagag
     2641 agaccacatg gtcttgttag aatttttgac tgctgctggt attaccgagg gtatgaatga
     2701 attgtacaaa tagtaagaat tctgcagata tccatcacac tggcggccgc tcgagcatgc
     2761 atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga
     2821 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2881 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2941 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3001 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3061 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3121 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3181 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3241 atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3301 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3361 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3421 accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt
     3481 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     3541 gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc
     3601 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3661 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3721 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3781 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3841 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3901 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3961 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4021 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4081 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     4141 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4201 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4261 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4321 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4381 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4441 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4501 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4561 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4621 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4681 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4741 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4801 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4861 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4921 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4981 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     5041 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5101 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5161 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5221 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5281 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5341 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5401 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5461 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5521 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5581 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5641 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     5701 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5761 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5821 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5881 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5941 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6001 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6061 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6121 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6181 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6241 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6301 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6361 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6421 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6481 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6541 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6601 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6661 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6721 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6781 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6841 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6901 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6961 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7021 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7081 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7141 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7201 tccccgaaaa gtgccacctg acgtc
//
