LOCUS       sequence-141332-        6678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hook domain of Hook3 in bacteria for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6678)
  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 6678)
  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 aa 1-160
FEATURES             Location/Qualifiers
     source          1..6678
                     /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             3594..3611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3664..3682)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3678..3725
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3762..4217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3849..3868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4059..4080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4310..5125)
                     /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(4310..5125)
                     /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     5033..5052
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5117..5136
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5247..5835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5736..5755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5989..6006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6021..6163
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6089..6108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6249..6271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6265..6456)
                     /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 cccttcctga tgtgaacctt attggggagc
     3421 attctgatgc agcagagctt ggaaggatgc ttcagctcat cttaggctgt gctgtgaact
     3481 gtgaacagaa gcaagagtac atccaagcca ttatgatgat ggaggaatct gttcaacatg
     3541 ttgtcatgac agccattcaa gagctgatga gtaaataagc ggccgcactc gagcaccacc
     3601 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     3661 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3721 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     3781 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3841 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3901 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3961 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4021 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4081 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4141 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4201 attaacgctt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4261 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     4321 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     4381 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     4441 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     4501 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     4561 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     4621 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     4681 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     4741 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     4801 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     4861 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     4921 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     4981 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     5041 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     5101 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     5161 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5221 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5281 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5341 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5401 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5461 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5521 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5581 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5641 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5701 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5761 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5821 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5881 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5941 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6001 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     6061 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6121 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     6181 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     6241 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6301 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     6361 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     6421 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     6481 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     6541 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     6601 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     6661 aggtgttcca cagggtag
//
