LOCUS       sequence-413054-        6419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of LRRK2 Armadillo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6419)
  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 6419)
  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-LRRK2 Armadillo K439E
FEATURES             Location/Qualifiers
     source          1..6419
                     /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      1876..1970
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2014..2673
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVDRMLNELQQYCDEWQGGA"
     primer_bind     complement(2080..2099)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2738..2824
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2917..3999)
                     /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     3961..3980
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4000..4077)
                     /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(4161..4183)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4269..4409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4324..4343
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4424..4441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4595..5183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4675..4694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5354..6145,6146..6214))
                     /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     5977..5996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6215..6319)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6387..6405
                     /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 acggatccat ggctagtggc
      181 agctgtcagg ggtgcgaaga ggacgaggaa actctgaaga agttgatagt caggctgaac
      241 aatgtccagg aaggaaaaca gatagaaacg ctggtccaaa tcctggagga tctgctggtg
      301 ttcacgtact ccgagcgcgc ctccaagtta tttcaaggca aaaatatcca tgtgcctctg
      361 ttgatcgtct tggactccta tatgagagtc gcgagtgtgc agcaggtggg ttggtcactt
      421 ctgtgcaaat taatagaagt ctgtccaggt acaatgcaaa gcttaatggg accccaggat
      481 gttggaaatg attgggaagt ccttggtgtt caccaattga ttcttaaaat gctaacagtt
      541 cataatgcca gtgtaaactt gtcagtgatt ggactgaaga ccttagatct cctcctaact
      601 tcaggtaaaa tcaccttgct gatattggat gaagaaagtg atattttcat gttaattttt
      661 gatgccatgc actcatttcc agccaatgat gaagtccaga aacttggatg caaagcttta
      721 catgtgctgt ttgagagagt ctcagaggag caactgactg aatttgttga gaacaaagat
      781 tatatgatat tgttaagtgc gttaacaaat tttaaagatg aagaggaaat tgtgcttcat
      841 gtgctgcatt gtttacattc cctagcgatt ccttgcaata atgtggaagt cctcatgagt
      901 ggcaatgtca ggtgttataa tattgtggtg gaagctatga aagcattccc tatgagtgaa
      961 agaattcaag aagtgagttg ctgtttgctc cataggctta cattaggtaa ttttttcaat
     1021 atcctggtat taaacgaagt ccatgagttt gtggtgaaag ctgtgcagca gtacccagag
     1081 aatgcagcat tgcagatctc agcgctcagc tgtttggccc tcctcactga gactattttc
     1141 ttaaatcaag atttagagga aaagaatgag aatcaagaga atgatgatga gggggaagaa
     1201 gataaattgt tttggctgga agcctgttac aaagcattaa cgtggcatag aaagaacaag
     1261 cacgtgcagg aggccgcatg ctgggcacta aataatctcc ttatgtacca aaacagttta
     1321 catgagaaga ttggagatga agatggccat ttcccagctc atagggaagt gatgctctcc
     1381 atgctgatgc attcttcatc aaaggaagtt ttccaggcat ctgcgaatgc attgtcaact
     1441 ctcttagaac aaaatgttaa tttcagaaaa atactgttat cagaaggaat acacctgaat
     1501 gttttggagt taatgcagaa gcatatacat tctcctgaag tggctgaaag tggctgtaaa
     1561 atgctaaatc atctttttga aggaagcaac acttccctgg atataatggc agcagtggtc
     1621 cccaaaatac taacagttat gaaacgtcat gagacatcat taccagtgca gctggaggcg
     1681 cttcgagcta ttttacattt tatagtgcct ggcatgccag aagaatccag ggaggataca
     1741 gaatttcatc ataagctaaa tatggttaaa aaacagtgtt tcaagaatga tattcacaaa
     1801 ctggtcctag cagctttgaa caggtagtct agaattccct gcaggtaatt aaataagctt
     1861 aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
     1921 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc
     1981 ttggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     2041 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     2101 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     2161 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     2221 catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     2281 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     2341 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     2401 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     2461 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     2521 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     2581 caggttcatc atgccgtttg tgatggcttc catgtcgaca gaatgcttaa tgaattacaa
     2641 cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct
     2701 taaacgcctg gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc
     2761 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     2821 aaatccgccc tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
     2881 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2941 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3001 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3061 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3121 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     3181 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     3241 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     3301 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     3361 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     3421 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     3481 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     3541 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     3601 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     3661 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     3721 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     3781 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     3841 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     3901 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     3961 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4021 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcgga
     4081 atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt
     4141 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4201 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4261 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     4321 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     4381 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     4441 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4501 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4561 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4621 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4681 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4741 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4801 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4861 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4921 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4981 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     5041 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5101 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5161 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5221 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5281 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5341 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5401 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5461 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5521 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5581 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5641 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5701 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5761 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5821 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5881 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5941 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6001 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6061 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6121 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6181 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6241 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6301 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6361 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
