LOCUS       sequence-346976-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Highly processive KIF1A for optogenetic heterodimerization to iLID 
            via SSPB(micro).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7901)
  AUTHORS   Nijenhuis W, van Grinsven MMP, Kapitein LC
  TITLE     An optimized toolbox for the optogenetic control of intracellular 
            transport.
  JOURNAL   J Cell Biol. 2020 Apr 6;219(4). pii: 133834. doi: 
            10.1083/jcb.201907149.
  PUBMED    32328628
REFERENCE   2  (bases 1 to 7901)
  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: "J Cell 
            Biol."; date: "2020-04-6"; volume: "219(4). pii: 133834. doi"; 
            pages: " 10.1083/jcb.201907149"
COMMENT     Sequence Label: pB80-KIF1A(1-365)-GCN4-GFP-SSPB(micro)
FEATURES             Location/Qualifiers
     source          1..7901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     583..605
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1650..1659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1916..1918,1919..2629)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1916..1918,1919..2629)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1958..1979)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2219..2238)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2566..2587
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(2759..2965,2966..2968,2969..3088)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    3221..3355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3258..3277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3312..3331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3369..3385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3369..3385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3382..3404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3393..3409
                     /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)."
     promoter        complement(join(3417..3423,3424..3441,3442..3447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3462..3483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3600..3617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3771..4359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3851..3870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4530..5321,5322..5390))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5153..5172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5391..5495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5563..5581
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5619..5641)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5741..5760
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5954..5976
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5968..5985
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5969..5985
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
       61 gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc
      121 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtgcggggc
      181 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
      241 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
      301 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
      361 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
      421 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
      481 agggccttcg tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgtc
      541 cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
      601 tgaccggcgg ggtttatatc ttcccttctc tgttcctccg cagcccccaa gctaggcaat
      661 tgggcgcgcc accatggccg gggcttcggt gaaggtggcg gtgcgtgtcc gccccttcaa
      721 ctcccgggaa atgagccgag actccaagtg catcattcag atgtctggaa gcaccaccac
      781 tattgtcaac cccaaacagc ccaaggagac acccaagagc ttcagctttg attactccta
      841 ctggtcacat acctcgccgg aggacatcaa ctatgcctct cagaagcagg tctacaggga
      901 cattggggaa gagatgctac agcatgcctt tgagggctac aatgtgtgca tctttgccta
      961 tgggcagacg ggggccggca agtcctacac catgatgggg aaacaggaga aggaccagca
     1021 aggcatcatt ccacagctct gcgaagacct cttctctcga atcaacgaca ccaccaacga
     1081 caacatgtcc tactctgtgg aggtcagcta catggagatt tactgtgagc gagtccgtga
     1141 cctcctgaac cccaagaaca aaggcaacct gcgtgtgagg gagcaccctc tgctggggcc
     1201 ttatgtggag gacctctcca agctggctgt aacctcctac aatgacatcc aggacctcat
     1261 ggactcaggg aacaaggcca ggactgtggc ggccacaaac atgaatgaga ccagcagccg
     1321 ttctcacgca gtcttcaaca tcatctttac ccagaagcgc catgatgcag agaccaacat
     1381 caccacagag aaagtgagca aaatcagcct ggtggacctg gcaggcagtg aacgagccga
     1441 ctccacagga gccaagggca cgcgactcaa ggagggggcc aacatcaaca agtctttgac
     1501 caccctggga aaggtcatct ctgctctggc tgaaatggac tctggaccca acaagaacaa
     1561 gaaaaagaag aagacagatt tcatccctta ccgagactcc gtattgacat ggcttctccg
     1621 ggaaaacctc ggtggcaact caaggacagc catggtggca gccctgagcc ctgcggacat
     1681 caactatgat gagaccctca gcacactgag atatgcagat cgggccaaac aaatccgctg
     1741 caatgccatc atcaatgaag accccaactc tagaggtggc gggggcagtg ggggatccct
     1801 tgaagacaag atcgaagaac tactttcgaa attgtaccac attgaaaatg agttggccag
     1861 acttaagaaa ttaatcggcg aaattgtcga cgggagcggg ggtgggagta agcttgtgag
     1921 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1981 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2041 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2101 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2161 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2221 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2281 cattgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2341 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2401 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2461 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2521 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2581 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg ctagcggagg
     2641 cggtggctca ggcggaggag ggtcaggagg gggaggcagt ggcgggggcg gctctggagg
     2701 agggggttct ggtggtggta gctacgtaga tctgagcggc ctgaccctgc aggaattcag
     2761 ctccccgaaa cgccctaagc tgctgcgtga atattacgat tggctggttg ataacagctt
     2821 taccccatat ctggtggtgg atgccacata cctgggcgtg aacgtgcccg tggagtatgt
     2881 gaaagacggt cagatcgtgc tgaatctgtc tgcaagtgcg accggcaacc tgcaactgac
     2941 aaatgatttt atccagttca acgcccagtt taagggcgtg tctcgtgaac tgtatatccc
     3001 gatgggtgcc gctctggcca tttacgctcg cgagaacggc gatggtgtga tgttcgaacc
     3061 agaagaaatc tatgacgagc tgaatattgg ttaaaccggt cctagataag taatgatcat
     3121 aatcagccat atcacatctg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3181 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3241 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3301 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaattc
     3361 gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3421 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3481 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3541 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3601 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3661 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3721 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3781 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3841 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3901 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3961 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4021 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4081 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4141 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4201 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4261 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4321 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4381 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4441 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4501 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4561 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4621 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4681 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4741 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4801 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4861 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4921 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4981 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5041 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5101 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5161 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5221 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5281 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5341 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5401 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5461 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5521 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5581 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     5641 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     5701 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     5761 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5821 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     5881 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     5941 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gttgggatct
     6001 ttgcattggc ccacggctct caggatgggg atgctcccct tcagcacccg gttccccttg
     6061 gaaactgatg gtcctggctc tgtggcatgg cagtggcact gtgaggagcc ccctaccagc
     6121 agcacacagt ggggttggca ctgccacgct ccggatgccg cgctctgatc caaccccata
     6181 atcaagggaa cccgaattgc cccatcattg cccccaccac ccccatcctg ccgggccctc
     6241 acaccccacg ctgccttctg gtgacattcc ccagcccaaa cccacggctt catggctacc
     6301 gcggggcatt tcccattgcc gccccattat cagctctgca cacctcccgc tgtacccatg
     6361 cctcgtggct gcccttcttt gacgtataat cttctaatta atacccggcc ttgtcaaagt
     6421 ggagcacaaa cgttaattaa ttccccagca ggcaggtaat taacagtgtg actccctttt
     6481 tgctgcgagt ggggctgata cagagagatg tggcactatg gagcccacgg ggtcctggca
     6541 ctgggtgccc acggaggtcc ccatgtgctg cagtgtcacc gcctccgagg agacagtatt
     6601 gtccctgcgg tgtccctgca gctcagctct gtccacaggg ccacctccag tttggagggg
     6661 acacaatgca gccccgatgc aacccatcct cgcagcatcc cagggacaaa gaccccactg
     6721 caagaccgca cacagggctg ggtcccgctc ccctaatatc tacagtgctt cttgcatggc
     6781 cccttaatca atgcagttaa tcagcatgcg ctcatgcacc gctctggagc tgcaaagccc
     6841 ctcgcagcgc tgctcaccaa caccgcgcac cgccccggcc cagcctgcag cacgcgctgc
     6901 aaacaggaaa gaaacaaaat attgcccaaa tgtaggcaaa ggcattcggc tgccttgacc
     6961 tccgccgggc cgggccctgc ctgactcagc tccttactca gcgctcgctt cctccctccg
     7021 gctgccaccg ccgcagcgca caccctgaca aagagtggcc cttaacgggc tctgaggtgc
     7081 acccagcagt gcactcagca gtccaagggc cggcctggag gtttgcaccg ctacgtgctg
     7141 acattagcat tgaacttggc cctgggtagt gctgcaggcc gggcggggtg ggtgtagaga
     7201 gtgcagcgcg cgttgcaccc ggtgcccctt cccctccctt gcatcccagc aggctgcacc
     7261 ccagcaccag gcccgtgcat gcatgctcct ggtgttattg cagcctggtg catgcatgcg
     7321 tcttgatggt gcagcgctgt gcatgcatcc tccttggtgt gtagcagtct agtgcatgca
     7381 tacccctcgg tgttattgct gctctgtgca cgcacgctca ttgtatcact tcatcccagt
     7441 gcatgcactc acactggagc gattgctgct cggtgcacgc acactcattg tatcactgca
     7501 gctcagtggc tgcacgcaca ccggtgttat tgctgctcgg tgcgtgcatg cacatcagtg
     7561 tcgctgcagc tcagtgcatg cacgctcatt gcccatcgct atccctgcct ctcctgctgg
     7621 cgctccccgg gaggtgactt caaggggacc gcaggaccac ctcgggggtg gggggagggc
     7681 tgcacacgcg gaccccgctc cccctcccca acaaagcact gtggaatcaa aaagggggga
     7741 ggggggatgg aggggcgcgt cacacccccg ccccacaccc tcacctcgag gtgagcccca
     7801 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     7861 ttttttaatt attttgtgca gcgatggggg cggggggggg g
//
