LOCUS       sequence-388922-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for GST-based purification of 
            Drosophila melanogaster kinesin heavy chain [1-421] with N-terminal 
            SNAP tag and cleavable N-terminal GST.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  TITLE     Bacterial expression plasmids for the purification of labelled DmKHC
            for motor-PAINT
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6817)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGEX-6p-1_GST-SNAP-DmKHC[1-421]
FEATURES             Location/Qualifiers
     source          1..6817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             960..1502
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     CDS             1515..1535
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2867..2889)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3034..3052)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3120..3224
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3225..3293,3294..4085)
                     /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(3443..3462)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4256..4844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4745..4764
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4998..5015
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5088..5165
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5166..6248
                     /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(5185..5204)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6297..6302,6303..6320,6321..6327)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6335..6351
                     /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     6359..6375
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6359..6375
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6371..6817,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6391..6408)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6391..6407)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6400..6422)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccggaattcg
      961 acaaagactg cgaaatgaag cgcaccaccc tggatagccc tctgggcaag ctggaactgt
     1021 ctgggtgcga acagggcctg caccgtatca tcttcctggg caaaggaaca tctgccgccg
     1081 acgccgtgga agtgcctgcc ccagccgccg tgctgggcgg accagagcca ctgatgcagg
     1141 ccaccgcctg gctcaacgcc tactttcacc agcctgaggc catcgaggag ttccctgtgc
     1201 cagccctgca ccacccagtg ttccagcagg agagctttac ccgccaggtg ctgtggaaac
     1261 tgctgaaagt ggtgaagttc ggagaggtca tcagctacag ccacctggcc gccctggccg
     1321 gcaatcccgc cgccaccgcc gccgtgaaaa ccgccctgag cggaaatccc gtgcccattc
     1381 tgatcccctg ccaccgggtg gtgcagggcg acctggacgt ggggggctac gagggcgggc
     1441 tcgccgtgaa agagtggctg ctggcccacg agggccacag actgggcaag cctgggctgg
     1501 gtgagccaac cactgaggat ctgtactttc agagcgataa cagcgcagaa cgagaaattc
     1561 cagccgagga cagcatcaaa gtggtctgcc gattccgacc gctgaacgac agcgaagaga
     1621 aggccggctc caagttcgtg gtcaagttcc ccaacaatgt ggaggagaac tgcatatcca
     1681 tagcgggcaa ggtgtatttg ttcgacaagg tcttcaaacc gaatgcatcc caggaaaagg
     1741 tctacaatga ggcggccaag tccattgtta cggatgtcct ggccgggtac aatggaacga
     1801 tattcgcata cggtcagacg tcctccggaa aaacgcatac gatggagggc gtgatcgggg
     1861 actccgtaaa acagggtatc atacctagga tcgtcaacga cattttcaat cacatctacg
     1921 cgatggaggt gaacctggag tttcacatca aggtctccta ctacgagatc tacatggaca
     1981 agattcgaga tctgttggac gtctccaagg tgaaccttag tgtgcacgag gataagaacc
     2041 gggtgccgta cgtcaagggc gctacggaac ggttcgtctc gtcgccggag gatgttttcg
     2101 aggtgatcga ggagggcaaa tccaatcgtc acatcgctgt gacaaacatg aacgagcatt
     2161 cttcgcgatc ccactcagta ttccttatca atgtgaagca ggagaacctg gagaaccaga
     2221 agaaactatc cggcaaactc tacctggtgg atttggccgg atccgagaag gtttccaaga
     2281 ctggagcgga gggaaccgtt cttgatgaag ccaagaacat caacaagtcg ctgtcggcct
     2341 tgggcaacgt aatttctgcc ctggcggacg gaaacaaaac gcacatcccc taccgtgatt
     2401 ccaagctaac gcgcatcctg caggagtcgc tgggaggcaa cgcacgcaca accatcgtca
     2461 tctgctgctc tccagccagt ttcaacgaat ctgaaacgaa gtcaacgctg gacttcggtc
     2521 gtagagccaa gacagtgaag aacgtggtct gcgttaacga ggagcttact gccgaggaat
     2581 ggaagcgacg ctatgaaaag gagaaggaaa agaacgcccg actaaagggt aaggtggaga
     2641 agcttgagat cgagcttgcg cgctggagag cgggtgaaac tgttaaggcg gaggagcaaa
     2701 tcaacatgga ggatctcatg gaggcaagca cgcccaacct ggaagtggag gcagcacaga
     2761 cggcggcggc cgaggccgct ttggccgccc agcgaacggc ttaggagcgg ccgcatcgtg
     2821 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2881 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2941 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3001 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     3061 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3121 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3181 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3241 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3301 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3361 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3421 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3481 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3541 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3601 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3661 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3721 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     3781 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3841 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3901 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3961 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4021 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4081 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4141 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4201 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4261 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4321 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4381 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4441 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4501 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4561 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4621 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4681 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4741 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4801 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4861 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4921 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4981 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5041 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     5101 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     5161 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     5221 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     5281 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     5341 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     5401 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     5461 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     5521 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     5581 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     5641 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     5701 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     5761 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     5821 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5881 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5941 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     6001 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     6061 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     6121 tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgcaa accgcctctc
     6181 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     6241 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     6301 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     6361 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     6421 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     6481 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     6541 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     6601 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     6661 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     6721 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     6781 aggccagacg cgaattattt ttgatggcgt tggaatt
//
