LOCUS       sequence-225012-        6617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Halo-RagB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6617)
  AUTHORS   Lawrence RE, Cho KF, Rappold R, Thrun A, Tofaute M, Kim DJ, 
            Moldavski O, Hurley JH, Zoncu R
  TITLE     A nutrient-induced affinity switch controls mTORC1 activation by its
            Rag GTPase-Ragulator lysosomal scaffold.
  JOURNAL   Nat Cell Biol. 2018 Sep;20(9):1052-1063. doi: 
            10.1038/s41556-018-0148-6. Epub 2018 Jul 30.
  PUBMED    30061680
REFERENCE   2  (bases 1 to 6617)
  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: 
            "10.1038/s41556-018-0148-6"; journalName: "Nat Cell Biol"; date: 
            "2018-09"; volume: "20"; issue: "9"; pages: "1052-1063"
COMMENT     Sequence Label: pRK5 Halo RagB
FEATURES             Location/Qualifiers
     source          1..6617
                     /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      925..934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             931..1818
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             complement(1817..1834)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2917..2926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2930..3064
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2967..2986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3021..3040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3133..3490
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3133..3324
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3156..3176)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3341..3476
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3510..3527)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3510..3526)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3519..3541)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3739..4194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3826..3845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4036..4057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4211..4230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4330..4352
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4390..4408)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4476..4580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4581..4649,4650..5441)
                     /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(4799..4818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5612..6200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6101..6120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6354..6371
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6488..6509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6524..6529,6530..6547,6548..6554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6562..6578
                     /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     6567..6589
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6586..6602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6586..6602
                     /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 attcgccacc atggaaatcg gtactggctt tccattcgac
      961 ccccattatg tggaagtcct gggcgagcgc atgcactacg tagatgttgg tccgcgcgat
     1021 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
     1081 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
     1141 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
     1201 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
     1261 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
     1321 atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc
     1381 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
     1441 acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag
     1501 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     1561 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     1621 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     1681 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     1741 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtct
     1801 actctggaga tttccggtgg tggtggtggt ggtgcgtcga cgatggaaga atctgactct
     1861 gagaaaacga cggagaaaga aaatctgggg ccgagaatgg atccaccact aggggaaccg
     1921 gaaggatcgc ttgggtgggt gctaccaaat acagccatga agaaaaaggt gctgttgatg
     1981 ggtaaaagtg ggtctggtaa gaccagcatg aggtctatta tctttgcaaa ttatattgcc
     2041 agagacacac gtcgccttgg cgcaacaatt gatgtagaac attctcatgt tcgatttctg
     2101 ggaaacctgg tattgaacct gtgggattgt ggtgggcaag acaccttcat ggaaaattat
     2161 ttcactagcc aacgggacaa catcttccga aatgtggagg ttctgattta tgtctttgat
     2221 gtggagagcc gcgaactgga aaaggacatg cactattacc aatcatgcct ggaggccatt
     2281 ctgcagaatt ctccagatgc caaaatattt tgcttggtac acaaaatgga tctggtacag
     2341 gaggatcaac gggacctgat ttttaaagag cgagaagaag atttgaggcg tttgtctcgc
     2401 ccattggaat gttcttgttt ccgaacatct atctgggatg aaaccctcta taaggcttgg
     2461 tccagcatag tttatcagct gattcccaat gttcagcagc tggaaatgaa cctaaggaat
     2521 tttgctgaaa ttatcgaagc tgatgaagta cttctttttg agagagctac ttttctggta
     2581 atttctcact atcagtgtaa agagcagcgt gatgcccata gatttgagaa aataagcaac
     2641 attattaagc agttcaagct gagctgcagc aagctggctg cctctttcca gagtatggaa
     2701 gtcaggaact ctaacttcgc tgctttcatt gacatcttta catccaacac ttatgtgatg
     2761 gttgtgatgt ctgatccgtc cattccttct gcagctactc tgatcaacat ccgcaatgcc
     2821 aggaaacact ttgaaaagct ggaaagagtg gatggaccaa agcagtgtct tctcatgcgc
     2881 taagcggccg ctaagtaagt aaggatcccc agcttggccg ccatggccca acttgtttat
     2941 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3001 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3061 gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg
     3121 gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3181 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3241 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3301 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3361 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3421 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3481 cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg tcgtgactgg
     3541 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3601 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3661 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3721 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3781 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4081 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4141 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     4201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4261 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4321 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4381 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4441 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4501 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4561 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4621 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4681 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4741 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4801 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4861 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4921 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4981 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5041 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5101 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5161 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5221 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5281 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5341 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5401 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5521 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6301 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6361 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6481 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6541 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6601 acatgattac gaattaa
//
