LOCUS       sequence-139748-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7327)
  AUTHORS   Bar-Peled L, Chantranupong L, Cherniack AD, Chen WW, Ottina KA, 
            Grabiner BC, Spear ED, Carter SL, Meyerson M, Sabatini DM
  TITLE     A Tumor suppressor complex with GAP activity for the Rag GTPases 
            that signal amino acid sufficiency to mTORC1.
  JOURNAL   Science. 2013 May 31;340(6136):1100-6. doi: 10.1126/science.1232044.
  PUBMED    23723238
REFERENCE   2  (bases 1 to 7327)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2013-05-31- 31"; volume: "340"; 
            issue: "6136"; pages: "1100-6"
COMMENT     Sequence Label: C terminal Flag Mios pRK5
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(1942..1953)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3570..3593
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3614..3623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3627..3761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3664..3683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3718..3737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3830..4187
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3830..4021
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3853..3873)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4038..4173
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4207..4224)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4207..4223)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4216..4238)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4436..4891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4523..4542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4733..4754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4908..4927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5027..5049
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5087..5105)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5173..5277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5278..5346,5347..6138)
                     /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(5496..5515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6309..6897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6798..6817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7051..7068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7185..7206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7221..7226,7227..7244,7245..7251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7259..7275
                     /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     7264..7286
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7283..7299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7283..7299
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cgtcgacgcc accatgagcg gtaccaaacc tgatatttta tgggcaccac
      961 accatgttga tagatttgtt gtgtgtgact cagaactaag tctttatcat gtggaatcta
     1021 ctgtgaactc agaactcaaa gctggatctt tacgtttatc tgaagactct gcagctacat
     1081 tactgtcaat aaattcagat acaccctata tgaaatgtgt tgcctggtat cttaattatg
     1141 atcctgaatg tctgctggca gttggacaag caaatggtcg agttgtactt acaagccttg
     1201 gtcaagatca taactcaaag ttcaaagatt tgataggaaa agagtttgtt ccaaaacatg
     1261 cacgacaatg taataccctt gcctggaatc cactggatag taactggcta gctgctggtt
     1321 tagataagca cagagctgac ttttcagtgc taatatggga tatctgcagc aaatatactc
     1381 ctgatatagt tcccatggaa aaagtgaaac tttcagcagg tgaaactgaa acaacattat
     1441 tagtaacaaa accactttat gagttaggac agaatgatgc ttgtctgtct ctttgttggc
     1501 ttccacgaga ccagaaactt ctccttgctg gtatgcatcg taacctagct atatttgatc
     1561 ttcggaatac aagccaaaag atgttcgtaa atacaaaagc tgttcagggt gtgacggtag
     1621 acccatattt ccacgatcgt gttgcttcct tctatgaagg tcaggttgca atatgggatc
     1681 ttagaaaatt tgagaagcca gttttgacat tgactgagca accaaaaccc ttaacaaaag
     1741 tagcatggtg tcccactagg actggtctac ttgccacttt aacaagggat agtaatatta
     1801 ttagattgta tgatatgcag catacaccca ctcccattgg ggatgaaact gaacccacaa
     1861 taattgaaag aagtgtgcaa ccttgtgaca attacattgc ttcctttgcg tggcatccaa
     1921 caagtcaaaa tcgaatgata gttgtaactc ccaaccgaac aatgtcagac ttcactgttt
     1981 ttgaaaggat atctcttgcc tggagcccaa ttacatcttt aatgtgggct tgtggtcgtc
     2041 atttatatga atgtacggaa gaagaaaatg ataattcttt agaaaaagat atagcaacga
     2101 agatgcgtct tcgggcttta tcaaggtatg gacttgatac agagcaggtg tggaggaacc
     2161 acattttagc tggaaatgaa gatccacagc tcaagtcact ctggtatact ctgcacttta
     2221 tgaagcaata cacagaagat atggatcaga aatctccagg caacaaagga tcattggttt
     2281 atgcaggaat taaatcaatt gtaaagtcat cgttgggaat ggtggaaagc agcagacata
     2341 attggagtgg gttggataag caaagtgata ttcaaaattt aaatgaagag agaatcttag
     2401 ctttacagct ttgtgggtgg ataaagaaag gaacggatgt agacgtgggg ccatttttga
     2461 actcccttgt acaagaaggg gaatgggaaa gagctgctgc tgtggcattg ttcaacttgg
     2521 atattcgccg agcaatccaa atcctgaatg aaggggcatc ttctgaaaaa ggagatctga
     2581 atctcaatgt ggtagcaatg gctttatcgg gttatacgga tgagaagaac tccctttgga
     2641 gagaaatgtg tagcacactg cgattacagc taaataaccc gtatttgtgt gtcatgtttg
     2701 catttctgac aagtgaaaca ggatcttacg atggagtttt gtatgaaaac aaagttgcag
     2761 tacgtgacag agtggcattt gcttgtaaat tccttagtga tactcagtta aatagataca
     2821 tcgaaaagtt gaccaatgaa atgaaagagg ctggaaattt ggaaggaatt ttgcttacag
     2881 gccttactaa agatggagtg gacttaatgg agagttatgt tgatagaact ggagatgttc
     2941 aaacagcaag ttactgtatg ttacagggtt cacctttaga tgttcttaaa gatgaaaggg
     3001 ttcagtactg gattgagaat tatagaaatt tattagatgc ctggaggttt tggcataaac
     3061 gagctgaatt tgatattcac aggagtaagt tggatcccag ttccaagcct ttagcacaag
     3121 tttttgtgag ttgcaatttc tgtggcaagt caatctccta cagctgttca gctgtgcctc
     3181 atcagggcag aggttttagt cagtatggtg tgagtggctc accaacgaaa tctaaagtca
     3241 caagttgtcc tggctgtcga aaaccacttc ctcgatgtgc gctttgtctc attaatatgg
     3301 gaacaccagt ttctagctgt cctggaggaa ccaaatcaga tgaaaaagtg gacttgagca
     3361 aggacaaaaa attagcccaa tttaacaact ggtttacatg gtgtcataat tgcaggcacg
     3421 gtggacatgc tggacatatg cttagttggt tcagggacca tgcagagtgc cctgtgtctg
     3481 catgcacgtg taaatgtatg cagttggata caacggggaa tctggtacct gcagagactg
     3541 tccagccagc ggccgcagga ggtggaggtg actacaagga tgacgatgac aagtgataag
     3601 gatccccagc ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat
     3661 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3721 gtttgtccaa actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag
     3781 caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa
     3841 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3901 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3961 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4021 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4081 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4141 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag
     4201 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4261 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4321 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     4381 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4441 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4501 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4561 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4621 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4681 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4741 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     4801 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4861 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4921 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4981 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5041 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5101 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5281 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5341 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5401 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5461 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5521 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5581 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5641 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5701 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5761 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5821 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5881 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5941 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6001 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6061 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6121 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6181 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6241 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6301 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6361 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6421 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6481 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6541 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6601 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6661 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6721 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6781 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6841 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6901 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6961 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7021 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7081 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     7141 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     7201 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     7261 gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa ttaattcgag
     7321 ctcgccc
//
