LOCUS       sequence-366230-        5354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2xHis-Rab29.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5354)
  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 5354)
  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-Rab29
FEATURES             Location/Qualifiers
     source          1..5354
                     /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             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      811..905
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             949..1608
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1015..1034)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1673..1759
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1852..2934)
                     /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     2896..2915
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2935..3012)
                     /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(3096..3118)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3204..3344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3259..3278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3359..3376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3530..4118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3610..3629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4289..5080,5081..5149))
                     /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     4912..4931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5150..5254)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5322..5340
                     /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 aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc atgggcagcc gcgaccacct gttcaaagtg
      181 ctggtggtgg gggacgccgc agtgggcaag acgtcgctgg tgcagcgata ttcccaggac
      241 agcttcagca aacactacaa gtccacggtg ggagtggatt ttgctctgaa ggttctccag
      301 tggtctgact acgagatagt gcggcttcag ctgtgggata ttgcagggca ggagcgcttc
      361 acctctatga cacgattgta ttatcgggat gcctctgcct gtgttattat gtttgacgtt
      421 accaatgcca ctaccttcag caacagccag aggtggaaac aggacctaga cagcaagctc
      481 acactaccca atggagagcc ggtgccctgc ctgctcttgg ccaacaagtg tgatctgtcc
      541 ccttgggcag tgagccggga ccagattgac cggttcagta aagagaacgg tttcacaggt
      601 tggacagaaa catcagtcaa ggagaacaaa aatattaatg aggctatgag agtcctcatt
      661 gaaaagatga tgagaaattc cacagaagat atcatgtctt tgtccaccca aggggactac
      721 atcaatctac aaaccaagtc ctccagctgg tcctgctgct agtctagaat tccctgcagg
      781 taattaaata agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
      841 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
      901 agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
      961 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     1021 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     1081 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     1141 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     1201 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     1261 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     1321 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     1381 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     1441 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     1501 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     1561 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc
     1621 agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc atcaaataaa
     1681 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1741 tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa
     1801 catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1861 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1921 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1981 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     2041 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     2101 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     2161 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     2221 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     2281 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2341 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2401 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2461 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2521 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2581 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2641 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2701 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2761 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2821 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2881 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2941 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca
     3001 ttcgatggtg tcggaatttc gggcagcgtt gggtcctggc cacgggtgcg catgatctag
     3061 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3121 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3181 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     3241 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     3301 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3361 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3421 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3961 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4081 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4141 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4201 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4261 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4321 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4381 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4441 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4501 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4561 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4621 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4681 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4741 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4801 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4861 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4921 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4981 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5041 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5101 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5161 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5221 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5281 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5341 cctttcgtct tcac
//
