LOCUS       sequence-366229-        5240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2xHis-LRRK2 Armadillo 1-159.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5240)
  AUTHORS   Vides EG, Adhikari A, Chiang CY, Lis P, Purlyte E, Limouse C, 
            Shumate JL, Spinola-Lasso E, Dhekne HS, Alessi DR, Pfeffer SR
  TITLE     A feed-forward pathway drives LRRK2 kinase membrane recruitment and 
            activation.
  JOURNAL   Elife. 2022 Sep 23;11:e79771. doi: 10.7554/eLife.79771.
  PUBMED    36149401
REFERENCE   2  (bases 1 to 5240)
  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: "2022-09-23"; volume: "11:e79771. doi"; pages: " 
            10.7554/eLife.79771"
COMMENT     Sequence Label: pQE-80L 2xHis-LRRK2 Armadillo 1-159
FEATURES             Location/Qualifiers
     source          1..5240
                     /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      697..791
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             835..1494
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(901..920)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1559..1645
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1738..2820)
                     /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     2782..2801
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2821..2898)
                     /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(2982..3004)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3090..3230
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3145..3164
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3245..3262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3416..4004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3496..3515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4175..4966,4967..5035))
                     /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     4798..4817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5036..5140)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5208..5226
                     /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 ccgagcacgc 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 atatttagtc tagaattccc
      661 tgcaggtaat taaataagct taattagctg agcttggact cctgttgata gatccagtaa
      721 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
      781 ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
      841 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
      901 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      961 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     1021 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     1081 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     1141 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1201 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1261 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1321 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1381 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     1441 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     1501 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     1561 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1621 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     1681 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1741 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1801 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1861 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1921 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1981 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     2041 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     2101 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     2161 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2221 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2281 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2341 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2401 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2461 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2521 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2581 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2641 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2701 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2761 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2821 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2881 gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
     2941 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3001 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3061 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     3121 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     3181 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3841 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5041 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5101 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5161 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5221 cgaggccctt tcgtcttcac
//
