LOCUS       sequence-366164-        8023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human KIF1C with R169W mutation and GFP 
            tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8023)
  AUTHORS   Siddiqui N, Roth D, Toleikis A, Zwetsloot AJ, Cross RA, Straube A
  TITLE     Force generation of KIF1C is impaired by pathogenic mutations.
  JOURNAL   Curr Biol. 2022 Sep 12;32(17):3862-3870.e6. doi: 
            10.1016/j.cub.2022.07.029. Epub 2022 Aug 11.
  PUBMED    35961316
REFERENCE   2  (bases 1 to 8023)
  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: 
            "10.1016/j.cub.2022.07.029"; journalName: "Curr Biol"; date: 
            "2022-09-12- 12"; volume: "32"; issue: "17"; pages: "3862-3870.e6"
COMMENT     Sequence Label: pKIF1C_R169W-GFP
FEATURES             Location/Qualifiers
     source          1..8023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             102..896
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             102..896
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(156..175)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     766..785
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1084..1103)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1128..1175
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1504..2092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1993..2012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2267..2570
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2571..2774
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2725..2745
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5071..5080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6169..6178
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6175..6177,6178..6180,6181..6894)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6175..6177,6178..6180,6181..6891)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6220..6241)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6481..6500)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6828..6849
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    7017..7138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7054..7073)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7108..7127
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(7145..7600)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7282..7303)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7494..7513
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7627..7731
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(7733..8023,1..67)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        7733..7924
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(7756..7776)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(7941..8023,1..53)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
       61 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
      121 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
      181 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
      241 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
      301 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
      361 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
      421 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
      481 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
      541 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
      601 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
      661 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
      721 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
      781 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
      841 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
      901 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
      961 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     1021 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     1081 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     1141 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     1201 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     1261 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     1321 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     1381 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1441 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1501 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1561 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1621 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     1681 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1741 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1801 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1861 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1921 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1981 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2041 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2101 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2161 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt
     2221 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     2281 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     2341 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     2401 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     2461 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     2521 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     2581 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     2641 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     2701 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     2761 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg actcagatct
     2821 cgagctcaag cttcgaattc tggagctatg gctggtgcct cggtgaaagt ggcagtgagg
     2881 gttcggccct ttaacgcccg tgagaccagc caggatgcca agtgtgtggt cagcatgcag
     2941 ggcaacacca cctccatcat caatcctaaa cagagcaagg atgcccccaa aagcttcacc
     3001 tttgactact cctactggtc acacacttcg acggaggacc cccagtttgc atctcagcag
     3061 caagtgtatc gggacatcgg agaagagatg ctgctccacg cctttgaagg ctacaacgtg
     3121 tgcatctttg cctatgggca gaccggggct gggaaatcct ataccatgat ggggcgacag
     3181 gagccagggc agcagggcat cgtgccccag ctctgtgagg acctcttctc tcgcgttagt
     3241 gagaaccaga gtgctcagct atcctactct gtggaggtgt cttacatgga aatctactgt
     3301 gagcgggtac gagacctctt gaaccccaag agtcggggtt ctctgcgggt ctgggagcac
     3361 cccatcctgg gcccgtacgt gcaggacctg tccaaattgg ctgtgacctc ctacgcagac
     3421 attgctgacc tcatggactg tggaaataaa gcacggactg tggctgccac caacatgaat
     3481 gagaccagca gccgttccca tgccgtcttt accatcgtct tcacacagcg ctgccatgac
     3541 cagctcacgg ggctggactc ggagaaggtc agtaagatca gtttggtgga ccttgctggg
     3601 agtgagcgag ccgactcctc aggggcccgg ggcatgcgcc tgaaggaagg agccaacatc
     3661 aataagtccc tgactacact agggaaagtg atctcggccc ttgcagatat gcaatcaaag
     3721 aagcgaaagt cggattttat cccctacagg gactctgtgc tcacctggct gctcaaggaa
     3781 aatttggggg ggaactcacg cacagccatg attgcagccc tgagccctgc tgacatcaat
     3841 tacgaggaga ctctcagcac cctcaggtat gctgaccgca ccaagcaaat ccgctgcaat
     3901 gccatcatca acgaggaccc taatgcccgg ctgattagag agctgcagga ggaagtagcc
     3961 cggctgcggg aactgctgat ggctcaggga ctgtcagcct ctgctctgga aggcctgaag
     4021 acggaagaag ggagtgtcag aggcgccctg ccagctgtgt catctccccc agctccagtt
     4081 tcaccctcat cacccaccac acataatggg gagctggagc cgtcattctc ccccaacacg
     4141 gagtcccaga ttgggcctga ggaagccatg gagaggctgc aggagacaga gaagattata
     4201 gctgagctga acgagacatg ggaggagaag ctacgcaaga cagaagccct gaggatggag
     4261 agagaagcat tgctggctga gatgggggtg gccgtccggg aggatggggg aactgtgggc
     4321 gtcttctctc caaagaagac tccccacctg gtgaacctga acgaagaccc tctgatgtct
     4381 gagtgtctgc tctaccacat caaagatggc gtcaccaggg tcggccaagt agatatggac
     4441 atcaagctga ccggacagtt cattcgggag caacactgtc tgttccggag catcccccag
     4501 ccagatggag aagtggtggt cactctggag ccttgtgaag gagctgagac atatgtgaat
     4561 gggaagcttg tgacggagcc gctggtgctg aagtcaggga ataggattgt gatgggcaag
     4621 aaccacgttt tccgcttcaa ccacccggag caggcaaggc tggaacggga acgaggggtc
     4681 cccccacccc caggaccgcc ctctgagcca gtcgactgga actttgccca gaaggaactg
     4741 ctggagcagc aaggcatcga cataaagctg gaaatggaga agaggctgca ggatctggag
     4801 aatcagtacc ggaaagaaaa ggaagaagcc gatcttctgc tggagcagca gcgactgtat
     4861 gcagactcgg acagcgggga tgactctgac aagcgctctt gtgaagagag ctggaggctc
     4921 atctcctcct tgcgggagca gctgccgccc accacggtcc agaccattgt caaacgctgt
     4981 ggtctgccca gcagtggcaa gcgcagggcc cctcgcaggg tttatcagat cccccagcga
     5041 cgcaggctgc agggcaaaga cccccgctgg gccaccatgg ctgacctgaa gatgcaggcg
     5101 gtgaaggaga tctgctacga ggtggccctg gctgacttcc gccacgggcg ggctgagatt
     5161 gaggccctgg ccgccctcaa gatgcgggag ctgtgtcgca cctatggcaa gccagacggc
     5221 cccggagacg cctggagggc tgtggcccgg gatgtctggg acactgtagg cgaggaggaa
     5281 ggaggtggag ctggcagtgg tggtggcagt gaggagggag cccgaggggc ggaggtggag
     5341 gacctccggg cccacatcga caagctgacg gggattctgc aggaggtgaa gctgcagaac
     5401 agcagcaagg accgggagct gcaggccctg cgggaccgca tgctccgcat ggagagggtc
     5461 atccccctgg cccaggatca tgaggatgag aatgaagaag gtggtgaggt cccctgggcc
     5521 ccgcctgaag gatcagaggc agcagaggag gcagccccca gtgaccgcat gccgtcagcc
     5581 cggcccccct cgccaccact gtcaagctgg gagcgggtgt cacggctcat ggaggaggac
     5641 cctgccttcc gtcgtggtcg tcttcgctgg ctcaagcagg agcagctacg gctgcaggga
     5701 ctgcagggct ctgggggccg gggcgggggg ctgcgcaggc ccccagcccg ctttgtgccc
     5761 cctcacgact gcaagctacg cttccccttc aagagcaacc cccagcaccg ggagtcttgg
     5821 ccagggatgg ggagcgggga ggctccaact ccgctccaac cccctgagga ggtcactccc
     5881 catccagcca cccctgcccg ccggcctccg agtccccgaa ggtcccacca tccccgcagg
     5941 aactccctgg atggaggggg ccgatcccgg ggagcgggtt ctgcacagcc tgaaccccag
     6001 cacttccagc ccaaaaagca caactcttat ccccagccac cccaacccta cccagcccag
     6061 cggcccccag ggccccgcta ccccccatac actactcccc cacgaatgag acggcagcgt
     6121 tctgcccctg acctcaagga gagtggggca gctgtggatc caccggtcgc caccatggtg
     6181 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     6241 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     6301 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     6361 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     6421 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     6481 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     6541 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     6601 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     6661 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     6721 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     6781 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     6841 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc
     6901 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     6961 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     7021 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     7081 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     7141 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     7201 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     7261 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     7321 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     7381 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     7441 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     7501 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     7561 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     7621 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7681 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     7741 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     7801 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     7861 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     7921 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     7981 cccatggctg actaattttt tttatttatg cagaggccga ggc
//
