LOCUS       sequence-459455-        5851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His-tagged Rab8A in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5851)
  AUTHORS   Raig ND, Surridge KJ, Sanz-Murillo M, Dederer V, Kramer A, Schwalm 
            MP, Elson L, Chatterjee D, Mathea S, Hanke T, Leschziner AE, 
            Reck-Peterson SL, Knapp S
  TITLE     Type-II kinase inhibitors that target Parkinson's Disease-associated
            LRRK2.
  JOURNAL   bioRxiv [Preprint]. 2025 Feb 13:2024.09.17.613365. doi: 
            10.1101/2024.09.17.613365.
  PUBMED    39554022
REFERENCE   2  (bases 1 to 5851)
  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: "bioRxiv 
            [Preprint]. 2025 Feb 13:2024.09.17.613365. doi: 
            10.1101/2024.09.17.613365."
COMMENT     Sequence Label: pET28A-Rab8A
FEATURES             Location/Qualifiers
     source          1..5851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(726..746)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(756..773)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             792..814
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    829..853
                     /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)."
     primer_bind     complement(853..872)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(854..872)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(956..975)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1181..1258
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1259..2341
                     /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     complement(1278..1297)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3150..3341
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3335..3357)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3443..3582
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3498..3517
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3597..3614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3768..4356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3848..3867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4467..4486)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4478..5293
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4478..5293
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4551..4570)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5385..5840)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5522..5543)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5734..5753
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttatttt ttgtccattt tcgccttgat gtcacgcgcc
      241 agggtaaaga acgcattttc cacattaatg ttcgccttcg cgctggtttc catgaatttg
      301 atgccataat ccagcgccag cttttcacca cgctctttgc taacctgacg cttatcgttc
      361 acgtcgcact tgttgcccag aatcattttt tcaacgtccg cgctcgcgtg ttcctcaatg
      421 ttacgaatcc agttacggat gttatcaaag ctcttctcgt tggtaatgtc gtacaccagc
      481 atgatgccca tcgcaccacg atagtacgcg gtggtaatgg tacggaaacg ttcttgaccc
      541 gcggtgtccc aaatctgcag tttgatacgc ttgccatcca gctcaatggt acggatttta
      601 aagtcaatac cgatggtgct gatgaaggtg ctgttgaacg catcctcgct aaaacggaac
      661 agaacgcagg tcttgcccac accgctatcg ccaatcagca gcagcttgaa caggtagtcc
      721 atatggccct gaaaataaag attctcgccg ctgctgtgat gatgatgatg atggctgctg
      781 cccatggtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
      841 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc
      901 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
      961 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     1021 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     1081 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     1141 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     1201 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     1261 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     1321 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     1381 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     1441 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     1501 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     1561 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     1621 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     1681 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     1741 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     1801 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     1861 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     1921 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     1981 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     2041 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     2101 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     2161 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     2221 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     2281 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     2341 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     2401 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     2461 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     2521 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     2581 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     2641 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg
     2701 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     2761 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     2821 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     2881 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     2941 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     3001 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
     3061 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
     3121 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
     3181 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     3241 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     3301 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     3361 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     3421 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     3481 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt
     3541 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     3601 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3661 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3721 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3781 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3841 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3901 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3961 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4021 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4081 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4141 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4201 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4261 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4321 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4381 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     4441 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     4501 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     4561 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     4621 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     4681 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     4741 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     4801 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     4861 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     4921 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     4981 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     5041 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     5101 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     5161 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     5221 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     5281 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     5401 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5461 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5521 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5581 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5641 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5701 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5761 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     5821 tgcgccgcta cagggcgcgt cccattcgcc a
//
