LOCUS       sequence-388919-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for GST-based purification of 
            Drosophila melanogaster kinesin heavy chain [1-421] with C-terminal 
            GFP tag and cleavable N-terminal GST tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  TITLE     Bacterial expression plasmids for the purification of labelled DmKHC
            for motor-PAINT
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6933)
  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            "Unpublished"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-DmKHC[1-421]-1xGFP
FEATURES             Location/Qualifiers
     source          1..6933
                     /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
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGSCPKERAEISMLEGAVLDIRY
                     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             join(2220..2222,2223..2225,2226..2939)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2220..2222,2223..2225,2226..2936)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2265..2286)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2526..2545)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2873..2894
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2983..3005)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3150..3168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3236..3340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3341..3409,3410..4201)
                     /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(3559..3578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4372..4960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4861..4880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5114..5131
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5204..5281
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5282..6364
                     /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(5301..5320)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6413..6418,6419..6436,6437..6443)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6451..6467
                     /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     6475..6491
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6475..6491
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6487..6933,1..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(6507..6524)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6507..6523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6516..6538)
                     /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 ttgggtagtt 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 atgagcgcag
      961 aacgagaaat tccagccgag gacagcatca aagtggtctg ccgattccga ccgctgaacg
     1021 acagcgaaga gaaggccggc tccaagttcg tggtcaagtt ccccaacaat gtggaggaga
     1081 actgcatatc catagcgggc aaggtgtatt tgttcgacaa ggtcttcaaa ccgaatgcat
     1141 cccaggaaaa ggtctacaat gaggcggcca agtccattgt tacggatgtc ctggccgggt
     1201 acaatggaac gatattcgca tacggtcaga cgtcctccgg aaaaacgcat acgatggagg
     1261 gcgtgatcgg ggactccgta aaacagggta tcatacctag gatcgtcaac gacattttca
     1321 atcacatcta cgcgatggag gtgaacctgg agtttcacat caaggtctcc tactacgaga
     1381 tctacatgga caagattcga gatctgttgg acgtctccaa ggtgaacctt agtgtgcacg
     1441 aggataagaa ccgggtgccg tacgtcaagg gcgctacgga acggttcgtc tcgtcgccgg
     1501 aggatgtttt cgaggtgatc gaggagggca aatccaatcg tcacatcgct gtgacaaaca
     1561 tgaacgagca ttcttcgcga tcccactcag tattccttat caatgtgaag caggagaacc
     1621 tggagaacca gaagaaacta tccggcaaac tctacctggt ggatttggcc ggatccgaga
     1681 aggtttccaa gactggagcg gagggaaccg ttcttgatga agccaagaac atcaacaagt
     1741 cgctgtcggc cttgggcaac gtaatttctg ccctggcgga cggaaacaaa acgcacatcc
     1801 cctaccgtga ttccaagcta acgcgcatcc tgcaggagtc gctgggaggc aacgcacgca
     1861 caaccatcgt catctgctgc tctccagcca gtttcaacga atctgaaacg aagtcaacgc
     1921 tggacttcgg tcgtagagcc aagacagtga agaacgtggt ctgcgttaac gaggagctta
     1981 ctgccgagga atggaagcga cgctatgaaa aggagaagga aaagaacgcc cgactaaagg
     2041 gtaaggtgga gaagcttgag atcgagcttg cgcgctggag agcgggtgaa actgttaagg
     2101 cggaggagca aatcaacatg gaggatctca tggaggcaag cacgcccaac ctggaagtgg
     2161 aggcagcaca gacggcggcg gccgaggccg ctttggccgc ccagcgaacg gctgaattca
     2221 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     2281 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2341 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2401 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     2461 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2521 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2581 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2641 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2701 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2761 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2821 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2881 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgac
     2941 tcgagcgatc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3001 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3061 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3121 cggagtgtat aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3181 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3241 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3301 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3361 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3421 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3481 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3541 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     3601 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3661 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3721 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3781 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3841 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     3901 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3961 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4021 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4081 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4141 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4201 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4261 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4321 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4381 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4441 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4501 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4561 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4621 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4681 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4741 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4801 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4861 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4921 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4981 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5041 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5101 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     5161 tctccttacg catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggcg
     5221 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     5281 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     5341 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     5401 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     5461 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     5521 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     5581 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     5641 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     5701 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     5761 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     5821 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     5881 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     5941 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     6001 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     6061 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     6121 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     6181 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     6241 cgtctcactg gtgaaaagaa aaaccacgct agcgcccaat acgcaaaccg cctctccccg
     6301 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6361 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact
     6421 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     6481 acagctatga ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa
     6541 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     6601 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     6661 tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat
     6721 cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg
     6781 cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag
     6841 aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc
     6901 cagacgcgaa ttatttttga tggcgttgga att
//
