LOCUS       sequence-471032-        5185 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 5185)
  AUTHORS   Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM
  TITLE     Ragulator Is a GEF for the Rag GTPases that Signal Amino Acid Levels
            to mTORC1.
  JOURNAL   Cell. 2012 Sep 14;150(6):1196-208. doi: 10.1016/j.cell.2012.07.032.
  PUBMED    22980980
REFERENCE   2  (bases 1 to 5185)
  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.2012.07"; journalName: "Cell"; date: "2012-09-14- 
            14"; volume: "150"; issue: "6"; pages: "1196-208"
COMMENT     Sequence Label: pRK5-p18-FLAG
FEATURES             Location/Qualifiers
     source          1..5185
                     /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"
     regulatory      918..927
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1428..1451
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1472..1481
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1485..1619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1522..1541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1576..1595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1688..2045
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1688..1879
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1711..1731)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1896..2031
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2065..2082)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2065..2081)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2074..2096)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2294..2749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2381..2400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2591..2612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2766..2785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2885..2907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2945..2963)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3031..3135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3136..3204,3205..3996)
                     /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(3354..3373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4167..4755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4656..4675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4909..4926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5043..5064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5079..5084,5085..5102,5103..5109)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5117..5133
                     /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     5122..5144
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5141..5157
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5141..5157
                     /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 accatggggt gctgctacag cagcgagaac gaggactcgg
      961 accaggaccg agaggagcgg aagctgctgc tggaccctag cagcccccct accaaagctc
     1021 tcaatggagc cgagcccaac taccacagcc tgccttccgc tcgcactgat gagcaggccc
     1081 tgctctcttc catccttgcc aagacagcca gcaacatcat tgatgtgtct gctgcagact
     1141 cacagggcat ggagcagcat gagtacatgg accgtgccag gcagtacagc acccgcttgg
     1201 ctgtgctgag cagcagcctg acccattgga agaagctgcc accgctgccg tctcttacca
     1261 gccagcccca ccaagtgctg gccagtgagc ccatcccgtt ctctgatttg cagcaggtct
     1321 ccaggatagc tgcttatgcc tacagtgcac tttctcagat ccgtgtggac gcaaaagagg
     1381 agctggttgt acagtttggg atcccagcgg ccgcaggagg tggaggtgac tacaaggatg
     1441 acgatgacaa gtgataagga tccccagctt ggccgccatg gcccaacttg tttattgcag
     1501 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1561 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     1621 ggaattaatt cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     1681 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     1741 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1801 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1861 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     1921 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     1981 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2041 gcaaaaagct gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2101 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2161 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2221 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     2281 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2341 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2401 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2461 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     2521 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2581 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     2641 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2701 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     2761 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2821 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2881 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     2941 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     3001 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3061 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3121 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3181 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3241 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3301 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3361 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3421 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3481 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3541 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3601 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3661 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3721 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3781 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3841 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3901 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3961 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4021 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4081 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4141 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4201 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4261 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4321 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4381 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4441 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4501 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4561 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4621 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4681 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4741 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4801 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4861 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4921 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4981 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     5041 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     5101 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     5161 attacgaatt aattcgagct cgccc
//
