LOCUS       sequence-405020-        5140 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 5140)
  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 5140)
  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-HA-mp1
FEATURES             Location/Qualifiers
     source          1..5140
                     /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"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1440..1449
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1453..1587
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1490..1509)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1544..1563
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1656..2013
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1656..1847
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1679..1699)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1864..1999
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2033..2050)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2033..2049)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2042..2064)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2262..2717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2349..2368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2559..2580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2734..2753)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2853..2875
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2913..2931)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2999..3103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3104..3172,3173..3964)
                     /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(3322..3341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4135..4723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4624..4643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4877..4894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5011..5032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5047..5052,5053..5070,5071..5077)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5085..5101
                     /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     5090..5112
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5109..5125
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5109..5125
                     /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 attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatggcggat gacctaaagc gattcttgta taaaaagtta ccaagtgttg
     1081 aagggctcca tgccattgtt gtgtcagata gagatggagt acctgttatt aaagtggcaa
     1141 atgacaatgc tccagagcat gctttgcgac ctggtttctt atccactttt gcccttgcaa
     1201 cagaccaagg aagcaaactt ggactttcca aaaataaaag tatcatctgt tactataaca
     1261 cctaccaggt ggttcaattt aatcgtttac ctttggtggt gagtttcata gccagcagca
     1321 gtgccaatac aggactaatt gtcagcctag aaaaggaact tgctccattg tttgaagaac
     1381 tgagacaagt tgtggaagtt tcttaagcgg ccgctaagta agtaaggatc cccagcttgg
     1441 ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1501 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1561 atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa
     1621 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg
     1681 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1741 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     1801 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     1861 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     1921 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     1981 ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg
     2041 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2101 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2161 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2221 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     2281 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2341 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2401 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2461 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2521 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2581 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     2641 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2701 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2761 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2821 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2881 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     2941 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3001 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3061 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3121 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3181 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3241 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3301 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3361 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3421 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3481 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3541 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3601 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3661 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3721 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3781 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3841 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3901 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3961 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4021 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4081 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4141 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4201 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4261 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4321 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4381 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4441 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4501 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4561 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4621 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4681 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4741 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4801 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4861 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4921 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4981 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     5041 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     5101 atttcacaca ggaaacagct atgacatgat tacgaattaa
//
