LOCUS       sequence-388923-        6924 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 
            mNeonGreen tag and cleavable N-terminal GST tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  AUTHORS   Chen F, Wu J, Iwanski MK, Jurriens D, Sandron A, Pasolli M, Puma G, 
            Kromhout JZ, Yang C, Nijenhuis W, Kapitein LC, Berger F, Akhmanova A
  TITLE     Self-assembly of pericentriolar material in interphase cells lacking
            centrioles.
  JOURNAL   Elife. 2022 Jul 5;11:e77892. doi: 10.7554/eLife.77892.
  PUBMED    35787744
REFERENCE   2  (bases 1 to 6924)
  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: "2022-07-5"; volume: "11:e77892. doi"; pages: " 
            10.7554/eLife.77892"
COMMENT     Sequence Label: pGEX-6p-1_GST-DmKHC[1-421]_1xmNeonGreen
FEATURES             Location/Qualifiers
     source          1..6924
                     /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             2220..2930
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2475..2484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2974..2996)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3141..3159)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3227..3331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3332..3400,3401..4192)
                     /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(3550..3569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4363..4951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4852..4871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5105..5122
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5195..5272
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5273..6355
                     /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(5292..5311)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6404..6409,6410..6427,6428..6434)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6442..6458
                     /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     6466..6482
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6466..6482
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6478..6924,1..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(6498..6515)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6498..6514)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6507..6529)
                     /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 gataacatgg cctctctccc agcgacacat gagttacaca
     2281 tctttggctc catcaacggt gtggactttg acatggtggg tcagggcacc ggcaatccaa
     2341 atgatggtta tgaggagtta aacctgaagt ccaccaaggg tgacctccag ttctccccct
     2401 ggattctggt ccctcatatc gggtatggct tccatcagta cctgccctac cctgacggga
     2461 tgtcgccttt ccaggccgcc atggtagatg gctccggata ccaagtccat cgcacaatgc
     2521 agtttgaaga tggtgcctcc cttactgtta actaccgcta cacctacgag ggaagccaca
     2581 tcaaaggaga ggcccaggtg aaggggactg gtttccctgc tgacggtcct gtgatgacca
     2641 actcgctgac cgctgcggac tggtgcaggt cgaagaagac ttaccccaac gacaaaacca
     2701 tcatcagtac ctttaagtgg agttacacca ctggaaatgg caagcgctac cggagcactg
     2761 cgcggaccac ctacaccttt gccaagccaa tggcggctaa ctatctgaag aaccagccga
     2821 tgtacgtgtt ccgtaagacg gagctcaagc actccaagac cgagctcaac ttcaaggagt
     2881 ggcaaaaggc ctttaccgat gtgatgggca tggacgagct gtacaagtga ctcgagcgat
     2941 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3001 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3061 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3121 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3181 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3241 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3301 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3361 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3421 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3481 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3541 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     3601 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3661 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3721 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3781 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3841 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     3901 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3961 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4021 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4081 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4141 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4201 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4261 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4321 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4381 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4441 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4501 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4561 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4621 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4681 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4741 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4801 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4861 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4921 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4981 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5041 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5101 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5161 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt
     5221 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     5281 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     5341 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     5401 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     5461 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     5521 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     5581 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     5641 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     5701 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     5761 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     5821 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     5881 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     5941 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     6001 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     6061 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     6121 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     6181 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     6241 ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6301 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6361 acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6421 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6481 accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     6541 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     6601 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt
     6661 gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag
     6721 gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac
     6781 accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg
     6841 ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga
     6901 attatttttg atggcgttgg aatt
//
