LOCUS       sequence-334927-        4677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kinesin KIF5B K490 cysteine light mutant with E215C mutation for 
            single cysteine labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4677)
  AUTHORS   Niekamp S, Sung J, Huynh W, Bhabha G, Vale RD, Stuurman N
  TITLE     Nanometer-accuracy distance measurements between fluorophores at the
            single-molecule level.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Mar 5;116(10):4275-4284. doi: 
            10.1073/pnas.1815826116. Epub 2019 Feb 15.
  PUBMED    30770448
REFERENCE   2  (bases 1 to 4677)
  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: 
            "10.1073/pnas.1815826116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-03-5- 5"; volume: "116"; issue: "10"; pages: "4275-4284"
COMMENT     Sequence Label: pET-17b_K490clm_E215C
FEATURES             Location/Qualifiers
     source          1..4677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1557..1574
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1635..1653)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1649..1696
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1741..1759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1827..1931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1932..2000,2001..2792)
                     /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(2150..2169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2963..3551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3452..3471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3705..3722
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3737..3876
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3802..3821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3962..3984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3978..4169)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atggcggacc tggccgagtc taatattaaa gtgatgtgtc
      121 gcttcagacc tctcaacgag tctgaagtga accgcggcga caagtacatc gccaagtttc
      181 agggagaaga cacggtcgtg atcgcgtcca agccttatgc atttgatcgg gtgttccagt
      241 caagcacatc tcaagagcaa gtgtataatg acgccgcaaa gaagattgtt aaagacgtcc
      301 ttgaaggata taatggaaca atatttgcat acggccaaac atcctctggg aagacacaca
      361 caatggaggg taaacttcat gatccagaag gcatgggaat tattccaaga atagtgcaag
      421 atatttttaa ttatatttac tccatggatg aaaatttgga atttcatatt aaggtttcat
      481 attttgaaat atatttggat aagataaggg acctgttaga tgtttcaaag accaaccttt
      541 cagttcatga agacaaaaac cgagttccct atgtaaaggg agccacagag cgttttgtaa
      601 gtagtccaga tgaagttatg gatacaatag atgaaggaaa atccaacaga catgtagcag
      661 ttacaaatat gaatgaacat agctctagga gtcacagtat atttcttatt aatgtcaaac
      721 aatgcaacac acaaacggaa caaaagctta gtggaaaact ttatctagtt gatttagctg
      781 gtagtgaaaa ggttagtaaa actggagctg aaggtgctgt gctggatgaa gctaaaaaca
      841 tcaacaagtc actttctgct cttggaaatg ttatttctgc tttggctgag ggtagtactt
      901 atgttccata tcgagatagt aaaatgacaa gaatccttca agatagccta ggtggcaacg
      961 ctagaaccac tattgtaatt tgctgctctc catcatcata caatgagtct gaaacaaaat
     1021 ctacactctt atttggccaa agggccaaaa caattaagaa cacagttagt gtcaatgtgg
     1081 agttaaccgc agaacagtgg aaaaagaagt atgaaaaaga aaaagaaaaa aataagatcc
     1141 tgcggaacac tattcagtgg cttgaaaatg agctcaacag atggcgtaat ggggagacgg
     1201 tgcctattga tgaacagttt gacaaagaga aagccaactt ggaagctttc acagtggata
     1261 aagatattac tcttaccaat gataaaccag caaccgcaat tggagttata ggcaatttta
     1321 ctgatgctga aagaagaaag gctgaagaag aaattgctaa attatacaaa cagcttgatg
     1381 acaaggatga agaaattaac cagcaaagtc aactggtaga gaaactgaag acgcaaatgt
     1441 tggatcagga ggagcttttg gcatctacca gaagggatca agacaatatg caagctgagc
     1501 tgaatcgcct tcaagcagaa aatgatgcct ctaaagaaga agtgaaagaa ggtacccatc
     1561 accatcatca ccattaactc gagcagatcc ggctgctaac aaagcccgaa aggaagctga
     1621 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1681 cttgaggggt tttttgctga aaggaggaac tatatccgga taattcttga agacgaaagg
     1741 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     1801 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1861 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1921 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1981 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2041 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2101 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2161 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2221 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2281 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2341 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2401 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2461 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2521 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2581 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2641 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2701 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2761 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2821 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2881 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2941 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3001 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3061 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3121 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3181 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3241 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3301 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3361 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3421 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3481 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3541 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3601 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3661 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3721 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3781 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3841 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3901 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3961 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4021 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4081 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4141 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4201 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4261 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4321 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4381 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4441 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4501 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4561 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4621 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaat
//
