LOCUS       sequence-416701-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-SUMO-tagged human Rab12 (Q101L).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  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 6698)
  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: pET 15b+His SUMO Rab12 (Q101L)
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(42..1124)
                     /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     1086..1105
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1125..1202)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1408..1427
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1511..1530
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1511..1529
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1530..1554
                     /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             1569..1591
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1604..1621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2709..2727)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2723..2770
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2945..2950,2951..2967,2968..2973))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3000..3018)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3086..3190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3191..3259,3260..4051)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3409..3428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4222..4810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4711..4730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4964..4981
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4996..5135
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5061..5080)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5221..5243
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5237..5428)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
       61 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      121 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
      181 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
      241 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
      301 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
      361 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
      421 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
      481 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
      541 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
      601 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      661 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      721 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      781 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      841 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      901 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      961 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     1021 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     1081 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     1141 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     1201 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1261 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     1321 cagtcccccg gccacggggc ctgccaccat acccacgcca aaacaagcgc tcatgagccc
     1381 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     1441 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     1501 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     1561 gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcatc accatcacca
     1621 ttctgaccag gaggcaaaac cttcaactga ggacttgggg gataagaagg aaggtgaata
     1681 tattaaactc aaagtcattg gacaggatag cagtgagatt cacttcaaag tgaaaatgac
     1741 aacacatctc aagaaactca aagaatcata ctgtcaaaga cagggtgttc caatgaactc
     1801 actcaggttt ctctttgagg gtcagagaat tgctgataat catactccaa aagaactggg
     1861 aatggaggaa gaagatgtga ttgaagttta tcaggaacaa acggggggat ccgatccggg
     1921 cgccgcgctg cagaggcggg ccgggggcgg cggcggtctg ggcgcgggct ccccggcgct
     1981 gtcgggcggc cagggccgcc ggaggaagca gccccccagg ccggccgact tcaagttgca
     2041 ggtcatcatt atcggctccc gcggcgtggg caagaccagc ctgatggagc gcttcaccga
     2101 cgacaccttc tgcgaggcct gcaagtccac cgtgggtgtt gacttcaaaa tcaaaactgt
     2161 agagctaaga ggaaagaaaa ttagattaca gatctgggac acagcaggtc tggagagatt
     2221 caacagcatt acctcagctt attacagaag tgccaagggg atcatattag tatatgatat
     2281 cactaagaag gagacatttg atgatttgcc gaaatggatg aagatgattg ataagtatgc
     2341 ttcagaagat gcagagcttc tcttagttgg aaataagttg gactgtgaaa cggacagaga
     2401 aatcaccagg cagcaggggg aaaagtttgc acagcagatc actgggatgc ggttctgtga
     2461 agcaagtgcc aaggataact tcaatgtgga cgagatattt ttgaaacttg tcgatgacat
     2521 tctgaaaaag atgcctctgg atattttaag gaatgagttg tccaatagta tcctgtcgtt
     2581 acaaccagag cctgagatac cgccagaact gcctccacca agaccacatg tccgatgctg
     2641 ttgatgacgg ccgcgtaaag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2701 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2761 gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc
     2821 ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc
     2881 gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta
     2941 ccgcattaaa gcttatcgat gataagctgt caaacatgag acttcttgaa gacgaaaggg
     3001 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3061 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3121 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3181 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3241 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3301 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3361 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3421 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3481 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3541 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3601 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3661 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3721 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3781 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3841 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3901 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3961 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4021 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4081 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4141 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4201 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4261 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4321 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4381 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4441 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4501 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4561 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4621 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4681 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4741 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4801 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4861 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4921 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4981 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5041 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     5101 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     5161 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5221 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     5281 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     5341 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     5401 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5461 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5521 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5581 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5641 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5701 acttccgcgt ttccagactt tacgaaacac gtaaaccgaa gaccattcat gttgttgctc
     5761 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5821 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     5881 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     5941 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     6001 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     6061 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     6121 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     6181 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     6241 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     6301 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     6361 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     6421 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     6481 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     6541 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     6601 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     6661 tgggttgaag gctctcaagg gcatcggtcg agatcccg
//
