LOCUS       sequence-141346-        6915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a double mutant truncation of Hook3 in bacteria for 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  AUTHORS   Schroeder CM, Vale RD
  TITLE     Assembly and activation of dynein-dynactin by the cargo adaptor 
            protein Hook3.
  JOURNAL   J Cell Biol. 2016 Aug 1;214(3):309-18. doi: 10.1083/jcb.201604002.
  PUBMED    27482052
REFERENCE   2  (bases 1 to 6915)
  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"; doi: "10.1083/jcb"; 
            journalName: "J Cell Biol"; date: "2016-08-1- 1"; volume: "214"; 
            issue: "3"; pages: "309-18"
COMMENT     Sequence Label: pET28a-Hook3 D102A, E108A
FEATURES             Location/Qualifiers
     source          1..6915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(587..1669)
                     /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     1631..1650
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1670..1747)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1953..1972
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2056..2075
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2056..2074
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2075..2099
                     /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)."
     RBS             2114..2136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2154..2171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2181..2198
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2202..2234
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(2250..2273,2274..2309,2310..2333)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             join(2340..2342,2343..3053)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2340..3053
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2370..2398)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2993..3013
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3060..3080
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3831..3848
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3901..3919)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3915..3962
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3999..4454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4086..4105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4296..4317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4547..5362)
                     /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             complement(4547..5362)
                     /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     5270..5289
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5354..5373
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5484..6072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5973..5992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6226..6243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6258..6400
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6326..6345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6486..6508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6502..6693)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
       61 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      121 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
      181 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
      241 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
      301 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
      361 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
      421 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
      481 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
      541 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
      601 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
      661 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
      721 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
      781 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      841 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
      901 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      961 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     1021 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     1081 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     1141 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     1201 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     1261 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     1321 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     1381 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     1441 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     1501 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     1561 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     1621 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     1681 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     1741 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     1801 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     1861 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     1921 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     1981 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     2041 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     2101 ctctagaaat aattttgttt aactttaaga aggagatata catgggcagc agccatcatc
     2161 atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatggctagc atgactggtg
     2221 gacagcaaat gggtcgcgga tccgaattct ggagtcatcc acaatttgag aagggcggcg
     2281 gcagtggcgg cggcagtggc ggcagtgcct ggagccaccc gcagttcgaa aaaggcagta
     2341 tgagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
     2401 atgttaatgg gcacaaattt tctgtccgtg gagagggtga aggtgatgct acaaacggaa
     2461 aactcaccct taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg
     2521 tcactactct gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc
     2581 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca
     2641 aagatgacgg gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta
     2701 atcgtatcga gttaaagggt attgatttta aagaagatgg aaacattctt ggacacaaac
     2761 tcgaatacaa ctttaactca cacaatgtat acatcacggc agacaaacaa aagaatggaa
     2821 tcaaagctaa cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc
     2881 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
     2941 tgtcgacaca atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc
     3001 ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac aaaggatccg
     3061 aaaatctgta ttttcagggg ggtggaggtg gtaccatgtt cagcgtagag tcgctggagc
     3121 gggcggagct gtgcgagagc ctcctcactt ggatccagac atttaatgtg gatgcaccat
     3181 gccagaccgt ggaagattta acgaatgggg ttgtgatggc ccaggttctt caaaagatag
     3241 atcctgcata ttttgatgaa aattggctaa acagaatcaa aactgaagta ggagataatt
     3301 ggaggctaaa gataagcaat ttaaagaaaa ttttaaaagg aatcttggat tataatcatg
     3361 agattttagg acagcaaatt aatgacttta cccttcctgc agtgaacctt attggggcac
     3421 attctgatgc agcagagctt ggaaggatgc ttcagctcat cttaggctgt gctgtgaact
     3481 gtgaacagaa gcaagagtac atccaagcca ttatgatgat ggaggaatct gttcaacatg
     3541 ttgtcatgac agccattcaa gagctgatga gtaaagaatc tcctgtctct gctggaaatg
     3601 atgcctatgt tgaccttgat cgtcagctga agaaaactac agaggaacta aatgaagctt
     3661 tgtcagcaaa ggaagaaatt gctcaaagat gccatgaact ggatatgcag gttgcagcat
     3721 tgcaggaaga gaaaagtagt ttgttggcag agaatcaggt attaatggaa agactcaatc
     3781 aatctgattc tatagaagac cctaacagtc cataagcggc cgcactcgag caccaccacc
     3841 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3901 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3961 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     4021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4141 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4441 aacgcttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4501 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     4561 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     4621 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     4681 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     4741 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     4801 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     4861 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     4921 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     4981 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     5041 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     5101 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     5161 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     5221 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     5281 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     5341 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     5401 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5461 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5521 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5581 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     5641 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5701 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5761 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5821 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5881 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5941 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6001 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6061 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6241 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     6301 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6361 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     6421 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6481 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     6541 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     6601 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     6661 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     6721 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     6781 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     6841 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     6901 tgttccacag ggtag
//
