LOCUS       sequence-163571-        5234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpression of CASTOR2 ACT1 domain (aa 1-171).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5234)
  AUTHORS   Chantranupong L, Scaria SM, Saxton RA, Gygi MP, Shen K, Wyant GA, 
            Wang T, Harper JW, Gygi SP, Sabatini DM
  TITLE     The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway.
  JOURNAL   Cell. 2016 Mar 24;165(1):153-64. doi: 10.1016/j.cell.2016.02.035. 
            Epub 2016 Mar 10.
  PUBMED    26972053
REFERENCE   2  (bases 1 to 5234)
  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.cell.2016.02.035"; journalName: "Cell"; date: 
            "2016-03-24- 24"; volume: "165"; issue: "1"; pages: "153-64"
COMMENT     Sequence Label: pRK5-Flag CASTOR2 ACT1
FEATURES             Location/Qualifiers
     source          1..5234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             937..960
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1534..1543
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1547..1681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1584..1603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1638..1657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1750..2107
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1750..1941
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1773..1793)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1958..2093
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2127..2144)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2127..2143)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2136..2158)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2356..2811
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2443..2462)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2653..2674
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2828..2847)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2947..2969
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3007..3025)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3093..3197
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3198..3266,3267..4058)
                     /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     complement(3416..3435)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4229..4817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4718..4737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4971..4988
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5105..5126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5141..5146,5147..5164,5165..5171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5179..5195
                     /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)."
     primer_bind     5184..5206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5203..5219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5203..5219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcaccatg gaaatggact acaaggatga cgatgacaag
      961 ggtggtggtg gtggtgcgtc gacgatggaa ctccacatcc tggagcaccg gctgcaagtt
     1021 gccagcgtcg ccaaggagag tatcccgctg ttcacctacg gcctgatcaa acttgccttc
     1081 ctgtcctcca agaccaggtg caagttcttc agtctgactg agacgccaga ggattacact
     1141 atcattgtcg atgaggaagg attcctagag ctgccctcct cggagcacct gagtgtggca
     1201 gatgccacct ggctggccct gaacgtggtg tccggcggtg gcagcttctc cagctcccag
     1261 cccatcggcg tgaccaagat cgccaagtca gtcatcgccc cactggctga ccagaacata
     1321 tccgtgttca tgctgtccac gtatcagaca gacttcatcc tggtgcgcga gcgggacctg
     1381 ccctttgtca cccacacatt gtcatcagag ttcaccatcc tgcgggtcgt caatggcgag
     1441 accgtggcag ccgagaacct cggcatcacc aatggcttcg tgaagcccaa gctggtctga
     1501 gcggccgcta agtaagtaag gatccccagc ttggccgcca tggcccaact tgtttattgc
     1561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     1681 cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     1741 aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     1801 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1861 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1921 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1981 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2041 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2101 ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa
     2161 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2221 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2281 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2341 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2401 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2461 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2521 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     2581 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2641 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     2701 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2761 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     2821 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     2881 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     2941 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3001 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3061 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3121 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3181 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3241 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3301 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3361 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3421 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3481 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3541 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3601 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3661 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3721 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3781 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3841 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3901 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3961 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4021 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4081 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4141 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4201 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4261 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4321 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4381 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4441 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4501 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4561 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4621 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4681 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4741 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4801 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4861 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4921 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4981 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5041 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     5101 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     5161 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca
     5221 tgattacgaa ttaa
//
