LOCUS       sequence-314042-        6503 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 6503)
  AUTHORS   Hsu PP, Kang SA, Rameseder J, Zhang Y, Ottina KA, Lim D, Peterson 
            TR, Choi Y, Gray NS, Yaffe MB, Marto JA, Sabatini DM
  TITLE     The mTOR-regulated phosphoproteome reveals a mechanism of 
            mTORC1-mediated inhibition of growth factor signaling.
  JOURNAL   Science. 2011 Jun 10;332(6035):1317-22.
  PUBMED    21659604
REFERENCE   2  (bases 1 to 6503)
  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: "Science."; 
            date: "2011-06-10"; volume: "332(6035)"; pages: "1317-22"
COMMENT     Sequence Label: pRK5 Flag Grb10 wt long
FEATURES             Location/Qualifiers
     source          1..6503
                     /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             924..947
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2790..2799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2803..2937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2840..2859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2894..2913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3006..3363
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3006..3197
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3029..3049)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3214..3349
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3383..3400)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3383..3399)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3392..3414)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3612..4067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3699..3718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3909..3930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4084..4103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4203..4225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4263..4281)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4349..4453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4454..4522,4523..5314)
                     /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(4672..4691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5485..6073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5974..5993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6227..6244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6361..6382
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6397..6402,6403..6420,6421..6427)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6435..6451
                     /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     6440..6462
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6459..6475
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6459..6475
                     /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 caccatggaa atggactaca aggatgacga tgacaagggt ggtggtggtg
      961 gtgcgtcgac gatggcttta gccggctgcc cagattcctt tttgcaccat ccgtactacc
     1021 aggacaaggt ggagcagaca cctcgcagtc aacaagaccc ggcaggacca ggactccccg
     1081 cacagtctga ccgacttgcg aatcaccagg aggatgatgt ggacctggaa gccctggtga
     1141 acgatatgaa tgcatccctg gagagcctgt actcggcctg cagcatgcag tcagacacgg
     1201 tgcccctcct gcagaatggc cagcatgccc gcagccagcc tcgggcttca ggccctcctc
     1261 ggtccatcca gccacaggtg tccccaaggc agagggtgca gcgctcccag cctgtgcaca
     1321 tcctcgctgt caggcgcctt caggaggaag accagcagtt tagaacctca tctctgccgg
     1381 ccatccccaa tccttttcct gaactctgtg gccctgggag cccccctgtg ctcacgccgg
     1441 gttctttacc tccgagccag gccgccgcaa agcaggatgt taaagtcttt agtgaagatg
     1501 ggacaagcaa agtggtggag attctagcag acatgacagc cagagacctg tgccaattgc
     1561 tggtttacaa aagtcactgt gtggatgaca acagctggac actagtggag caccacccgc
     1621 acctaggatt agagaggtgc ttggaagacc atgagctggt ggtccaggtg gagagtacca
     1681 tggccagtga gagtaaattt ctattcagga agaattacgc aaaatacgag ttctttaaaa
     1741 atcccatgaa tttcttccca gaacagatgg ttacttggtg ccagcagtca aatggcagtc
     1801 aaacccagct tttgcagaat tttctgaact ccagtagttg tcctgaaatt caagggtttt
     1861 tgcatgtgaa agagctggga aagaaatcat ggaaaaagct gtatgtgtgt ttgcggagat
     1921 ctggccttta ttgctccacc aagggaactt caaaggaacc cagacacctg cagctgctgg
     1981 ccgacctgga ggacagcaac atcttctccc tgatcgctgg caggaagcag tacaacgccc
     2041 ctacagacca cgggctctgc ataaagccaa acaaagtcag gaatgaaact aaagagctga
     2101 ggttgctctg tgcagaggac gagcaaacca ggacgtgctg gatgacagcg ttcagactcc
     2161 tcaagtatgg aatgctcctt taccagaatt accgaatccc tcagcagagg aaggccttgc
     2221 tgtccccgtt ctcgacgcca gtgcgcagtg tctccgagaa ctccctcgtg gcaatggatt
     2281 tttctgggca aacaggacgc gtgatagaga atccggcgga ggcccagagc gcagccctgg
     2341 aggagggcca cgcctggagg aagcgaagca cacggatgaa catcctaggt agccaaagtc
     2401 ccctccaccc ttctacccta agtacagtga ttcacaggac acagcactgg tttcacggga
     2461 ggatctccag ggaggaatcc cacaggatca ttaaacagca agggctcgtg gatgggcttt
     2521 ttctcctccg tgacagccag agtaatccaa aggcatttgt actcacactg tgtcatcacc
     2581 agaaaattaa aaatttccag atcttacctt gcgaggacga cgggcagacg ttcttcagcc
     2641 tagatgacgg gaacaccaaa ttctctgacc tgatccagct ggttgacttt taccagctga
     2701 acaaaggagt cctgccttgc aaactcaagc accactgcat ccgagtggcc ttatgagcgg
     2761 ccgctaagta agtaaggatc cccagcttgg ccgccatggc ccaacttgtt tattgcagct
     2821 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2881 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcggg
     2941 aattaattcg gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt
     3001 accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3061 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3121 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3181 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3241 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3301 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3361 aaaaagctgt taacagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     3421 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     3481 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     3541 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     3601 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3661 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3721 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3781 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     3841 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3901 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
     3961 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4021 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     4081 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4141 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4201 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4261 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4321 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4381 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4441 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4501 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4561 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4621 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4681 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     4741 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4801 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4861 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4921 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4981 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5041 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5101 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5161 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5221 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5281 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5341 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5401 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5461 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5521 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5581 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5641 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5701 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5761 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5821 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5881 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5941 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6001 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6061 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6121 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6181 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6241 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     6301 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     6361 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     6421 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
     6481 tacgaattaa ttcgagctcg ccc
//
