LOCUS       Exported                9915 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pPK121, complete sequence.
ACCESSION   AF055298
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9915)
  AUTHORS   Coy DL, Wagenbach M, Howard J.
  TITLE     Kinesin takes one 8-nm step for each ATP that it hydrolyzes
  JOURNAL   J. Biol. Chem. 274 (6), 3667-3671 (1999)
  PUBMED    9920916
REFERENCE   2  (bases 1 to 9915)
  AUTHORS   Coy DL, Wagenbach M, Howard J.
  TITLE     Mechanochemical Coupling Stoichiometry of Single Kinesin Motors
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 9915)
  AUTHORS   Coy DL, Wagenbach M, Howard J.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-MAR-1998) Physiology and Biophysics, University of 
            Washington, Campus Box 357290, Seattle, WA 98195-7290, USA
REFERENCE   4  (bases 1 to 9915)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9915
                     /organism="Expression vector pPK121"
                     /mol_type="other DNA"
                     /note="for expression of Drosophila melanogaster kinesin 
                     heavy chain and light chain in Escherichia coli"
                     /db_xref="taxon:74939"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        494..598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             599..1459
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1630..2218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2404..2546
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2648..2839)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3648..4730)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="LacI"
                     /protein_id="AAD13356.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3648..4730)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4731..4808)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        5117..5135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5117..5133
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     protein_bind    5136..5160
                     /label=lac repressor binding site
                     /bound_moiety="lac repressor"
                     /note="lac operator"
     RBS             5175..5197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5205..8132
                     /codon_start=1
                     /product="kinesin heavy chain"
                     /label=kinesin heavy chain
                     /note="derived from Drosophila melanogaster kinesin heavy 
                     chain, previously described in GenBank Accession Number 
                     M24441"
                     /protein_id="AAD13353.1"
                     /translation="MSAEREIPAEDSIKVVCRFRPLNDSEEKAGSKFVVKFPNNVEENC
                     ISIAGKVYLFDKVFKPNASQEKVYNEAAKSIVTDVLAGYNGTIFAYGQTSSGKTHTMEG
                     VIGDSVKQGIIPRIVNDIFNHIYAMEVNLEFHIKVSYYEIYMDKIRDLLDVSKVNLSVH
                     EDKNRVPYVKGATERFVSSPEDVFEVIEEGKSNRHIAVTNMNEHSSRSHSVFLINVKQE
                     NLENQKKLSGKLYLVDLAGSEKVSKTGAEGTVLDEAKNINKSLSALGNVISALADGNKT
                     HIPYRDSKLTRILQESLGGNARTTIVICCSPASFNESETKSTLDFGRRAKTVKNVVCVN
                     EELTAEEWKRRYEKEKEKNARLKGKVEKLEIELARWRAGETVKAEEQINMEDLMEASTP
                     NLEVEAAQTAAAEAALAAQRTALANMSASVAVNEQARLATECERLYQQLDDKDEEINQQ
                     SQYAEQLKEQVMEQEELIANARREYETLQSEMARIQQENESAKEEVKEVLQALEELTVN
                     YDQKSQEIDNKNKDIDALNEELQQKQSVFNAASTELQQLKDMSSHQKKRITEMLTNLLR
                     DLGEVGQAIAPGESSIDLKMSALAGTDASKVEEDFTMARLFISKMKTEAKNIAQRCSNM
                     ETQQADSNKKISEYEKDLGEYRLLISQHEARMKSLQESMREAENKKRTLEEQIDSLREE
                     CAKLKAAEHVSAVNAEEKQRAEELRSMFDSQMDELREAHTRQVSELRDEIAAKQHEMDE
                     MKDVHQKLLLAHQQMTADYEKVRQEDAEKSSELQNIILTNERREQARKDLKGLEDTVAK
                     ELQTLHNLRKLFVQDLQQRIRKNVVNEESEEDGGSLAQKQKISFLENNLDQLTKVHKQL
                     VRDNADLRCELPKLEKRLRCTMERVKALETALKEAKEGAMRDRKRYQYEVDRIKEAVRQ
                     KHLGRRGPQAQIAKPIRSGQGAIAIRGGGAVGGPSPLAQVNPVNS"
     RBS             6716..6724
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             8154..8177
                     /codon_start=1
                     /product="co-translated peptide"
                     /label=co-translated peptide
                     /note="modeled on Schoner, B.E. et al. (1984) PNAS USA 
                     81:5403-5407"
                     /protein_id="AAD13355.1"
                     /translation="MFPLEDD"
     CDS             8178..9752
                     /codon_start=1
                     /product="decahistidyl-kinesin light chain"
                     /label=decahistidyl-kinesin light chain
                     /note="derived from Drosophila melanogaster kinesin light 
                     chain, previously described in GenBank Accession Number 
                     L11328; initial 68 codons modified to improve codon usage 
                     and secondary structure of mRNA"
                     /protein_id="AAD13354.1"
                     /translation="MTQMSQDEIITNTKTVLQGLEALRVEHVSIMNGIAEVQKDNEKSD
                     MLRKNIENIELGLSEAQVMMALTSHLQNIEAEKHKLKTQVRRLHQENAWLRDELANTQQ
                     KFQASEQLVAQLEEEKKHLEFMASVKKYDENQEQDDACDKSRTDPVVELFPDEENEDRH
                     NMSPTPPSQFANQTSGYEIPARLRTLHNLVIQYASQGRYEVAVPLCKQALEDLERTSGH
                     DHPDVATMLNILALVYRDQNKYKEAANLLNDALSIRGKTLGENHPAVAATLNNLAVLYG
                     KRGKYKDAEPLCKRALEIREKVLGKDHPDVAKQLNNLALLCQNQGKYDEVEKYYQRALD
                     IYESKLGPDDPNVAKTKNNLAGCYLKQGRYTEAEILYKQVLTRAHEREFGAIDSKNKPI
                     WQVAEEREEHKFDNRENTPYGEYGGWHKAAKVDSPTVTTTLKNLGALYRRQGMFEAAET
                     LEDCAMRSKKEAYDLAKQTKLSQLLTSNEKRRSKAIKEDLDFSEEKNAKPLVPRGSHHH
                     HHHHHHH"
     CDS             9702..9719
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             9720..9749
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             9759..9776
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      9843..9890
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"