LOCUS       sequence-331194-        5338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of recombinant GST-R5BD (Rab5-binding domain) in bacteria
            for GST pulldown assays to identify binding partners for Rab5 
            subfamily members.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5338)
  AUTHORS   Qi Y, Liang Z, Wang Z, Lu G, Li G
  TITLE     Determination of Rab5 activity in the cell by effector pull-down 
            assay.
  JOURNAL   Methods Mol Biol. 2015;1298:259-70. doi: 
            10.1007/978-1-4939-2569-8_22.
  PUBMED    25800849
REFERENCE   2  (bases 1 to 5338)
  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: "Methods Mol
            Biol."; date: "2015"; volume: "1298:259-70. doi"; pages: " 
            10.1007/978-1-4939-2569-8_22"
COMMENT     Sequence Label: pGEX-4T-2/Rabaptin-5:R5BD
FEATURES             Location/Qualifiers
     source          1..5338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1206..1228)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1373..1391)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1459..1563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1564..1632,1633..2424)
                     /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(1782..1801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2595..3183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3084..3103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3337..3354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3427..3504
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3505..4587
                     /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(3524..3543)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4636..4641,4642..4659,4660..4666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4674..4690
                     /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     4698..4714
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4698..4714
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4710..5237
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4730..4747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4730..4746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4739..4761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tccccaggaa ttcgagcttc tatttctagc
      781 ctaaaagctg aattagaaag aataaaagtg gaaaaaggac agttggagtc cacattaaga
      841 gagaagtctc aacagcttga gagtcttcag gaaataaaga tcagtttgga agagcagtta
      901 aagaaagaga ctgctgctaa ggctaccgtt gaacagctaa tgtttgaaga gaagaacaaa
      961 gctcagagat tacagacaga attagatgtc agtgagcaag tccagagaga ttttgtaaag
     1021 ctttcacaga cccttcaggt gcagttagag cggatccggc aagctgactc cttggagaga
     1081 atccgggcaa ttctgaatga tactaaactg acagacatta accagcttcc tgagacatga
     1141 ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     1201 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     1261 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     1321 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     1381 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     1441 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1501 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1561 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1621 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1681 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1741 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1801 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1861 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1921 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1981 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2041 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2101 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2161 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2221 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2281 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2341 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2401 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2461 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     2521 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2581 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2641 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2701 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2761 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2821 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2881 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2941 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3001 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3061 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3121 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3181 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3241 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3301 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3361 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3421 aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     3481 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     3541 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     3601 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     3661 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     3721 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     3781 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     3841 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     3901 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     3961 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     4021 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     4081 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     4141 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     4201 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     4261 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     4321 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     4381 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     4441 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     4501 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4561 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     4621 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4681 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     4741 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4801 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4861 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     4921 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     4981 ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca
     5041 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     5101 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagct
     5161 tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     5221 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
     5281 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctg
//
