LOCUS       sequence-413055-        5492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Rab12.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5492)
  AUTHORS   Dhekne HS, Tonelli F, Yeshaw WM, Chiang CY, Limouse C, Jaimon E, 
            Purlyte E, Alessi DR, Pfeffer SR
  TITLE     Genome-wide screen reveals Rab12 GTPase as a critical activator of 
            Parkinson's disease-linked LRRK2 kinase.
  JOURNAL   Elife. 2023 Oct 24;12:e87098. doi: 10.7554/eLife.87098.
  PUBMED    37874635
REFERENCE   2  (bases 1 to 5492)
  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"; journalName: "Elife."; 
            date: "2023-10-24"; volume: "12:e87098. doi"; pages: " 
            10.7554/eLife.87098"
COMMENT     Sequence Label: pQE-80L 2xHis-Rab12 Q101L
FEATURES             Location/Qualifiers
     source          1..5492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             145..162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      949..1043
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1087..1746
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1153..1172)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1811..1897
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1990..3072)
                     /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
                     EDCKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3034..3053
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3073..3150)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3234..3256)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3342..3482
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3397..3416
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3497..3514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3668..4256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3748..3767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4427..5218,5219..5287))
                     /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     5050..5069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5288..5392)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5460..5478
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgcat
      121 caccatcacc atcacagagg atcgcatcac catcaccatc acggatccga tccgggcgcc
      181 gcgctgcaga ggcgggccgg gggcggcggc ggtctgggcg cgggctcccc ggcgctgtcg
      241 ggcggccagg gccgccggag gaagcagccc cccaggccgg ccgacttcaa gttgcaggtc
      301 atcattatcg gctcccgcgg cgtgggcaag accagcctga tggagcgctt caccgacgac
      361 accttctgcg aggcctgcaa gtccaccgtg ggtgttgact tcaaaatcaa aactgtagag
      421 ctaagaggaa agaaaattag attacagatc tgggacacag caggtctgga gagattcaac
      481 agcattacct cagcttatta cagaagtgcc aaggggatca tattagtata tgatatcact
      541 aagaaggaga catttgatga tttgccgaaa tggatgaaga tgattgataa gtatgcttca
      601 gaagatgcag agcttctctt agttggaaat aagttggact gtgaaacgga cagagaaatc
      661 accaggcagc agggggaaaa gtttgcacag cagatcactg ggatgcggtt ctgtgaagca
      721 agtgccaagg ataacttcaa tgtggacgag atatttttga aacttgtcga tgacattctg
      781 aaaaagatgc ctctggatat tttaaggaat gagttgtcca atagtatcct gtcgttacaa
      841 ccagagcctg agataccgcc agaactgcct ccaccaagac cacatgtccg atgctgttga
      901 tctagaattc cctgcaggta attaaataag cttaattagc tgagcttgga ctcctgttga
      961 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     1021 gggcgttttt tattggtgag aatccaagct agcttggcga gattttcagg agctaaggaa
     1081 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     1141 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     1201 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     1261 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aatttcgtat ggcaatgaaa
     1321 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     1381 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     1441 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     1501 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     1561 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     1621 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     1681 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
     1741 gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggggtaat gactctctag
     1801 cttgaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1861 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccctctagat tacgtgcagt
     1921 cgatgataag ctgtcaaaca tgagaattgt gcctaatgag tgagctaact tacattaatt
     1981 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2041 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     2101 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     2161 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     2221 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     2281 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     2341 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     2401 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     2461 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     2521 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     2581 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     2641 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     2701 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacagtcttc
     2761 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     2821 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     2881 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     2941 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     3001 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     3061 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     3121 gcgaaaggtt ttgcaccatt cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca
     3181 cgggtgcgca tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     3241 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3301 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     3361 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     3421 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3481 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3541 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3601 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3661 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3721 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3781 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3841 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3901 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3961 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4021 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4081 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4141 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4201 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4261 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4321 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4381 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4441 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4501 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4561 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4621 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4681 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4741 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4801 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4861 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4921 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4981 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5041 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5101 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5161 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5221 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5281 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5341 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5401 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5461 ataggcgtat cacgaggccc tttcgtcttc ac
//
