LOCUS       sequence-368136-        7603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged fusion protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7603)
  AUTHORS   Barutcu AR, Wu M, Braunschweig U, Dyakov BJA, Luo Z, Turner KM, 
            Durbic T, Lin ZY, Weatheritt RJ, Maass PG, Gingras AC, Blencowe BJ
  TITLE     Systematic mapping of nuclear domain-associated transcripts reveals 
            speckles and lamina as hubs of functionally distinct retained 
            introns.
  JOURNAL   Mol Cell. 2022 Mar 3;82(5):1035-1052.e9. doi: 
            10.1016/j.molcel.2021.12.010. Epub 2022 Feb 18.
  PUBMED    35182477
REFERENCE   2  (bases 1 to 7603)
  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: 
            "10.1016/j.molcel.2021.12.010"; journalName: "Mol Cell"; date: 
            "2022-03-3- 3"; volume: "82"; issue: "5"; pages: "1035-1052.e9"
COMMENT     Sequence Label: pDEST-cDNA5-FRT/TO-3*Flag-APEX2 C-term
FEATURES             Location/Qualifiers
     source          1..7603
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    987..1111
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1136..1141,1142..1159,1160..1166)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1220..1879
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1286..1305)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2221..2526
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2405..2424
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2567..2691)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2702..3448
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             3449..3514
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3555..3572)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3561..3785
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3918..3937)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4069..4116
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4124..5146
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5276..5397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5313..5332)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5367..5386
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5446..5462)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5446..5462)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5459..5481)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5470..5486
                     /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(5494..5500,5501..5518,5519..5524))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5539..5560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5677..5694)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5848..6436)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5928..5947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6607..7398,7399..7467))
                     /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     7230..7249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7468..7572)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag ctttagacaa gtttgtacaa aaaagctgaa cgagaaacgt
     1021 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
     1081 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
     1141 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
     1201 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     1261 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     1321 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     1381 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
     1441 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     1501 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     1561 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     1621 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     1681 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     1741 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     1801 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     1861 gtggcagggc ggggcgtaaa gatctggatc cggcttacta aaagccagat aacagtatgc
     1921 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
     1981 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
     2041 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
     2101 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
     2161 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
     2221 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     2281 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     2341 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     2401 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
     2461 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     2521 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat
     2581 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     2641 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg ttgattctag
     2701 aggaaagtct tacccaactg tgagtgctga ttaccaggac gccgttgaga aggcgaagaa
     2761 gaagctcaga ggcttcatcg ctgagaagag atgcgctcct ctaatgctcc gtttggcatt
     2821 ccactctgct ggaacctttg acaagggcac gaagaccggt ggacccttcg gaaccatcaa
     2881 gcaccctgcc gaactggctc acagcgctaa caacggtctt gacatcgctg ttaggctttt
     2941 ggagccactc aaggcggagt tccctatttt gagctacgcc gatttctacc agttggctgg
     3001 cgttgttgcc gttgaggtca cgggtggacc taaggttcca ttccaccctg gaagagagga
     3061 caagcctgag ccaccaccag agggtcgctt gcctgatccc actaagggtt ctgaccattt
     3121 gagagatgtg tttggcaaag ctatggggct tactgaccaa gatatcgttg ctctatctgg
     3181 gggtcacact attggagctg cacacaagga gcgttctgga tttgagggtc cctggacctc
     3241 taatcctctt attttcgaca actcatactt cacggagttg ttgagtggtg agaaggaagg
     3301 tctccttcag ctaccttctg acaaggctct tttgtctgac cctgtattcc gccctctcgt
     3361 tgataaatat gcagcggacg aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa
     3421 gctttccgag cttgggtttg ctgatgccga ctacaaagac catgacggtg attataaaga
     3481 tcatgacatc gattacaagg atgacgatga caagtaactc gagtctagag ggcccgttta
     3541 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3601 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3661 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3721 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3781 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3841 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3901 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3961 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4021 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     4081 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     4141 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     4201 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     4261 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     4321 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggagttca gcgagagcct
     4381 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     4441 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     4501 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     4561 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     4621 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     4681 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     4741 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     4801 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     4861 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     4921 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     4981 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     5041 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     5101 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     5161 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5221 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5281 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5341 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5401 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5461 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5521 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5581 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5641 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5701 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5761 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5821 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5881 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5941 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6001 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6061 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6121 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6181 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6241 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6301 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6361 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6421 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6481 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6541 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6601 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6661 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6721 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6781 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6841 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6901 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6961 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7021 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7081 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7141 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7201 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7261 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7321 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7381 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7441 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7501 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7561 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
