LOCUS       sequence-146232-        7356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used for expression in E. coli and purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7356)
  AUTHORS   Schroeder CM, Ostrem JM, Hertz NT, Vale RD
  TITLE     A Ras-like domain in the light intermediate chain bridges the dynein
            motor to a cargo-binding region.
  JOURNAL   Elife. 2014 Oct 1;3:e03351. doi: 10.7554/eLife.03351.
  PUBMED    25272277
REFERENCE   2  (bases 1 to 7356)
  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: "2014-10-1"; volume: "3:e03351. doi"; pages: " 
            10.7554/eLife.03351"
COMMENT     Sequence Label: pET28a-RILP
FEATURES             Location/Qualifiers
     source          1..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2412
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2338..2357)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2498..2520
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2514..2705)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3514..4596)
                     /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     4558..4577
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4597..4674)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4880..4899
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4983..5002
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4983..5001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5002..5026
                     /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             5041..5063
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5082..5099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5109..5126
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5133..5156,5157..5192,5193..5216)
                     /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(5223..5225,5226..5936)
                     /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             5223..5936
                     /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(5253..5281)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5876..5896
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             7158..7181
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7200..7217
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7270..7288)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7284..7331
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     2341 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2401 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2461 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2521 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2581 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     2641 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     2701 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     2761 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     2821 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     2881 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     2941 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3001 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3061 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3121 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     3181 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     3241 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     3301 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     3361 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     3421 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3481 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3541 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3601 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3661 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3721 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3781 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3841 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3901 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3961 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4021 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4081 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4141 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4201 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4261 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4321 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4381 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4441 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4501 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4561 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4621 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4681 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     4741 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     4801 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     4861 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4921 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     4981 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     5041 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
     5101 gcagcggcct ggtgccgcgc ggcagccata tgtggagtca tccacaattt gagaagggcg
     5161 gcggcagtgg cggcggcagt ggcggcagtg cctggagcca cccgcagttc gaaaaaggca
     5221 gtatgagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
     5281 gtgatgttaa tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat gctacaaacg
     5341 gaaaactcac ccttaaattt atttgcacta ctggaaaact acctgttccg tggccaacac
     5401 ttgtcactac tctgacctat ggtgttcaat gcttttcccg ttatccggat cacatgaaac
     5461 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt
     5521 tcaaagatga cgggacctac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg
     5581 ttaatcgtat cgagttaaag ggtattgatt ttaaagaaga tggaaacatt cttggacaca
     5641 aactcgaata caactttaac tcacacaatg tatacatcac ggcagacaaa caaaagaatg
     5701 gaatcaaagc taacttcaaa attcgccaca acgttgaaga tggttccgtt caactagcag
     5761 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     5821 acctgtcgac acaatctgtc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc
     5881 ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaaggat
     5941 ccgaattcat ggagcccagg agggcggcgc ccggggtgcc tggctggggg tctcgggagg
     6001 ccgcggggtc ggcatcggcc gcggagcttg tgtaccatct agccggggcc ctgggcactg
     6061 agctgcagga tctggcgcgc cgtttcgggc cggaggcggc ggccgggctg gtgccgctag
     6121 tggtgcgggc gctggagctc ttggaacagg ctgccgtggg gcccgccccg gactcgctgc
     6181 aggtgtcggc gcagccggcg gagcaggagc tgcggcggct gcgggaggag aacgagcgcc
     6241 tccgcaggga gctgcgcgcg gggccacagg aggagcgcgc gctgctgcgg cagctcaagg
     6301 aggtcacgga ccgacagcgg gacgaactcc gggcgcacaa ccgcgacctg cggcagcgcg
     6361 gccaggagac cgaggcgttg caggagcagc tgcagcgcct cctgctggtg aacgctgagc
     6421 tgcggcacaa gctggcggcc atgcagaccc agctgcgcgc cgcgcaggac cgcgagaggg
     6481 agcgccagca gcctggcgaa gccgcgactc cgcaggctaa agagcgagcg cgggggcagg
     6541 ccgggcggcc cgggcaccag cacggacagg agcccgaatg ggcgaccgcc ggcgcaggcg
     6601 ccccagggaa ccctgaggac ccggcggagg ccgcgcagca gctcgggcgc ccctcggagg
     6661 cagggcagtg ccgcttcagt cgggaggagt ttgagcagat ccttcaggag cggaatgaac
     6721 tcaaagccaa agtgttcctg ctcaaggagg aactggccta cttccagcgg gagctgctca
     6781 cagaccaccg ggtccccggc cttctgctcg aggccatgaa ggtggctgtc cggaagcagc
     6841 ggaagaagat caaggccaag atgttaggga caccagagga agcagagagc agtgaggatg
     6901 aggctggccc atggatcctg ctctccgatg acaagggaga ccatccccca cccccggagt
     6961 ccaaaataca gagtttcttt ggcctatggt atcggggtaa agctgaatcc tctgaggatg
     7021 agaccagcag ccctgcaccc agcaagctag ggggagaaga ggaggcccaa ccacagtctc
     7081 cagctcctga tccgccctgt tctgccctcc acgaacacct ttgtctgggg gcctcagccg
     7141 ccccagaggc cggtagcgat tacaaggatg acgacgataa gtaagcggcc gcactcgagc
     7201 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     7261 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     7321 ggggtttttt gctgaaagga ggaactatat ccggat
//
