LOCUS       sequence-273228-        6526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of GST-p18.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6526)
  AUTHORS   Shen K, Sabatini DM
  TITLE     Ragulator and SLC38A9 activate the Rag GTPases through noncanonical 
            GEF mechanisms.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9545-9550. doi: 
            10.1073/pnas.1811727115. Epub 2018 Sep 4.
  PUBMED    30181260
REFERENCE   2  (bases 1 to 6526)
  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: 
            "10.1073/pnas.1811727115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-09-18- 18"; volume: "115"; issue: "38"; pages: 
            "9545-9550"
COMMENT     Sequence Label: [pSK122] pETDuet GST-p18(delta5)
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             95..748
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     706..728
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             755..772
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        1323..1341
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1342..1366
                     /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)."
     CDS             1475..1519
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1557..1575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1571..1618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1655..2110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1742..1761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1952..1973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2225..3016,3017..3085))
                     /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     2848..2867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3086..3178)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3259..3847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3748..3767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4001..4018
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4033..4172
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4098..4117)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4258..4280
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4274..4465)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5037..6119)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6081..6100
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6120..6197)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6403..6422
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6510..6526,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca acactagtag caatatgtcc cctatactag gttattggaa
      121 aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga
      181 agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg
      241 tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat
      301 ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg
      361 tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat
      421 tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat
      481 gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac
      541 ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg
      601 cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat
      661 tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac
      721 gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt ccgcgtggat ccccgaattc
      781 gagcagcgag aacgaggact cggaccagga ccgagaggag cggaagctgc tgctggaccc
      841 tagcagcccc cctaccaaag ctctcaatgg agccgagccc aactaccaca gcctgccttc
      901 cgctcgcact gatgagcagg ccctgctctc ttccatcctt gccaagacag ccagcaacat
      961 cattgatgtg tctgctgcag actcacaggg catggagcag catgagtaca tggaccgtgc
     1021 caggcagtac agcacccgct tggctgtgct gagcagcagc ctgacccatt ggaagaagct
     1081 gccaccgctg ccgtctctta ccagccagcc ccaccaagtg ctggccagtg agcccatccc
     1141 gttctctgat ttgcagcagg tctccaggat agctgcttat gcctacagtg cactttctca
     1201 gatccgtgtg gacgcaaaag aggagctggt tgtacagttt gggatcccat gactcgagcg
     1261 gccgcataat gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa
     1321 attaatacga ctcactatag gggaattgtg agcggataac aattccccat cttagtatat
     1381 tagttaagta taagaaggag atatacatat ggcagatctc aattggatat cggccggcca
     1441 cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa accgctgctg cgaaatttga
     1501 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     1561 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1621 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1681 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1741 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1801 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1861 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1921 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1981 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2041 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2101 tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc
     2161 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2221 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2281 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2341 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2401 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2461 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2521 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2581 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2641 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2701 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2761 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2821 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2881 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2941 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3001 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3061 cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat
     3121 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3181 ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3241 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3301 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3361 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3421 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3481 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3541 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3601 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3661 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3721 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3781 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3841 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3901 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3961 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4021 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4081 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4141 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4201 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4261 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4321 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4381 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4441 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4501 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4561 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4621 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4681 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4741 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4801 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4861 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4921 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4981 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5101 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5161 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5221 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5281 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     5341 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5401 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5461 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5521 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5581 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5641 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5701 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5761 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5821 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5881 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5941 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6001 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6061 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6121 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6181 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6241 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     6301 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6361 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6421 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6481 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actata
//
