LOCUS       sequence-314049-        5096 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 5096)
  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 5096)
  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-FLAG-p14
FEATURES             Location/Qualifiers
     source          1..5096
                     /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      1383..1392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1396..1530
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1433..1452)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1487..1506
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1599..1956
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1599..1790
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1622..1642)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1807..1942
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1976..1993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1976..1992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1985..2007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2205..2660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2292..2311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2502..2523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2677..2696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2796..2818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2856..2874)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2942..3046
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3047..3115,3116..3907)
                     /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(3265..3284)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4078..4666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4567..4586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4820..4837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4954..4975
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4990..4995,4996..5013,5014..5020)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5028..5044
                     /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     5033..5055
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5052..5068
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5052..5068
                     /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 gatgctgcgc cccaaggctt tgacccaggt gctaagccaa gccaacactg
     1021 gaggcgtcca gagcaccctg ctgctgaata acgagggatc actgctggcc tactctggtt
     1081 acggggacac tgacgcccgg gtcaccgctg ccatagccag taacatctgg gccgcctacg
     1141 accggaacgg gaaccaagcg tttaatgaag acaatctcaa attcatcctc atggactgca
     1201 tggagggccg tgtagccatc acccgagtgg ccaaccttct gctgtgtatg tatgccaagg
     1261 agaccgtggg ctttggaatg ctcaaggcca aggcccaggc tttggtgcag tacctggagg
     1321 agcccctcac ccaagtggcg gcatcttaag cggccgctaa gtaagtaagg atccccagct
     1381 tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1441 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1501 ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct
     1561 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
     1621 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1681 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     1741 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     1801 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     1861 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     1921 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg
     1981 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2041 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2101 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     2161 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     2221 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2281 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2341 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2401 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2461 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2521 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     2581 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2641 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     2701 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2761 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2821 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     2881 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3001 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3061 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3121 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3181 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3241 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3301 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3361 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3421 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3481 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3541 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3601 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3661 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3721 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3781 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3841 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3901 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3961 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4021 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4081 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4141 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4201 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4261 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4321 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4381 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4441 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4501 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4561 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4621 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4681 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4741 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4801 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     4861 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4921 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     4981 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     5041 acaatttcac acaggaaaca gctatgacat gattacgaat taattcgagc tcgccc
//
