LOCUS       sequence-456536-        5240 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 5240)
  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 5240)
  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-HBXIP
FEATURES             Location/Qualifiers
     source          1..5240
                     /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"
     CDS             937..960
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1540..1549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1553..1687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1590..1609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1644..1663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1756..2113
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1756..1947
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1779..1799)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1964..2099
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2133..2150)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2133..2149)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2142..2164)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2362..2817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2449..2468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2659..2680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2834..2853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2953..2975
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3013..3031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3099..3203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3204..3272,3273..4064)
                     /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(3422..3441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4235..4823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4724..4743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4977..4994
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5111..5132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5147..5152,5153..5170,5171..5177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5201
                     /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     5190..5212
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5209..5225
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5209..5225
                     /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 attcaccatg gaaatggact acaaggatga cgatgacaag
      961 ggtggtggtg gtggtgcgtc gacgatggag ccaggtgcag gtcacctcga cggtcaccgc
     1021 gcggggagcc caagccttcg tcaggctctg tgcgacggaa gcgcagtgat gttttccagt
     1081 aaagaacgcg gacgttgcac cgtgatcaat tttgtccctt tggaggcgcc gttacggtcc
     1141 acgccccgct cgcgtcaagt gactgaggcc tgtggtggag aaggacgtgc cgtgccgctg
     1201 ggttctgagc cggagtggtc ggtgggtggg atggaggcga ccttggagca gcacttggaa
     1261 gacacaatga agaatccctc cattgttgga gtcctgtgca cagattcaca aggacttaat
     1321 ctgggttgcc gcgggaccct gtcagatgag catgctggag tgatatctgt tctagcccag
     1381 caagcagcta agctaacctc tgaccccact gatattcctg tggtgtgtct agaatcagat
     1441 aatgggaaca ttatgatcca gaaacacgat ggcatcacgg tggcagtgca caaaatggcc
     1501 tcttgagcgg ccgctaagta agtaaggatc cccagcttgg ccgccatggc ccaacttgtt
     1561 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     1621 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     1681 ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg aaagaggaac
     1741 ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     1801 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     1861 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1921 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     1981 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2041 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2101 aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca acgtcgtgac
     2161 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2221 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2281 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2341 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2401 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2461 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2521 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2581 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2641 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2701 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2761 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     2821 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     2881 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2941 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3001 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3061 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3181 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3421 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3721 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4381 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4981 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5041 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5101 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5161 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5221 atgacatgat tacgaattaa
//
