LOCUS       sequence-346993-        8318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Non-processive monomeric 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 8318)
  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 8318)
  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)-mVenus-SSPB(micro)
FEATURES             Location/Qualifiers
     source          1..8318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1048..1070
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1133..1142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2115..2124
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2333..2335,2336..3046)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2333..2335,2336..3046)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2375..2396)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2636..2655)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2983..3004
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(3176..3382,3383..3385,3386..3505)
                     /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    3638..3772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3675..3694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3729..3748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3786..3802)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3786..3802)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3799..3821)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3810..3826
                     /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(3834..3840,3841..3858,3859..3864))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3879..3900
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4017..4034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4188..4776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4268..4287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4947..5738,5739..5807))
                     /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     5570..5589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5808..5912)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5980..5998
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6036..6058)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6158..6177
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6371..6393
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6385..6402
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6386..6402
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
       61 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      121 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      181 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      241 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc
      301 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
      361 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
      421 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
      481 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
      541 agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc cctccccgag
      601 ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc gcggggctcg
      661 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
      721 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
      781 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
      841 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
      901 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
      961 cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgtccgcag ggggacggct
     1021 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggggttt
     1081 atatcttccc ttctctgttc ctccgcagcc cccaagctag gcaattgggc gcgccaccat
     1141 ggccggggct tcggtgaagg tggcggtgcg tgtccgcccc ttcaactccc gggaaatgag
     1201 ccgagactcc aagtgcatca ttcagatgtc tggaagcacc accactattg tcaaccccaa
     1261 acagcccaag gagacaccca agagcttcag ctttgattac tcctactggt cacatacctc
     1321 gccggaggac atcaactatg cctctcagaa gcaggtctac agggacattg gggaagagat
     1381 gctacagcat gcctttgagg gctacaatgt gtgcatcttt gcctatgggc agacgggggc
     1441 cggcaagtcc tacaccatga tggggaaaca ggagaaggac cagcaaggca tcattccaca
     1501 gctctgcgaa gacctcttct ctcgaatcaa cgacaccacc aacgacaaca tgtcctactc
     1561 tgtggaggtc agctacatgg agatttactg tgagcgagtc cgtgacctcc tgaaccccaa
     1621 gaacaaaggc aacctgcgtg tgagggagca ccctctgctg gggccttatg tggaggacct
     1681 ctccaagctg gctgtaacct cctacaatga catccaggac ctcatggact cagggaacaa
     1741 ggccaggact gtggcggcca caaacatgaa tgagaccagc agccgttctc acgcagtctt
     1801 caacatcatc tttacccaga agcgccatga tgcagagacc aacatcacca cagagaaagt
     1861 gagcaaaatc agcctggtgg acctggcagg cagtgaacga gccgactcca caggagccaa
     1921 gggcacgcga ctcaaggagg gggccaacat caacaagtct ttgaccaccc tgggaaaggt
     1981 catctctgct ctggctgaaa tggactctgg acccaacaag aacaagaaaa agaagaagac
     2041 agatttcatc ccttaccgag actccgtatt gacatggctt ctccgggaaa acctcggtgg
     2101 caactcaagg acagccatgg tggcagccct gagccctgcg gacatcaact atgatgagac
     2161 cctcagcaca ctgagatatg cagatcgggc caaacaaatc cgctgcaatg ccatcatcaa
     2221 tgaagacccc aactctagag gtggcggggg cagtggggga gggggaagtg gaggcggcgg
     2281 ttctggcggg ggaggctccg gtggcggggg gagcgggggt gggagtaagc ttgtgagcaa
     2341 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2401 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2461 cctgaagttg atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2521 cctcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     2581 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2641 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2701 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2761 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc
     2821 aaacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     2881 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     2941 ccagtccaaa ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3001 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggcta gcggaggcgg
     3061 tggctcaggc ggaggagggt caggaggggg aggcagtggc gggggcggct ctggaggagg
     3121 gggttctggt ggtggtagct acgtagatct gagcggcctg accctgcagg aattcagctc
     3181 cccgaaacgc cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac
     3241 cccatatctg gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa
     3301 agacggtcag atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa
     3361 tgattttatc cagttcaacg cccagtttaa gggcgtgtct cgtgaactgt atatcccgat
     3421 gggtgccgct ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga
     3481 agaaatctat gacgagctga atattggtta aaccggtcct agataagtaa tgatcataat
     3541 cagccatatc acatctgtag aggttttact tgctttaaaa aacctcccac acctccccct
     3601 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     3661 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3721 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaattcgta
     3781 atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3841 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3901 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3961 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4021 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4081 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4141 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4201 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4261 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4321 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4381 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4441 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4501 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4561 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4621 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4681 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4741 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4801 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4861 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4921 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4981 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5041 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5101 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5161 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5221 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5281 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5341 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5401 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5461 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5521 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5581 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5641 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5701 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5761 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5821 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5881 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5941 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     6001 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     6061 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     6121 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     6181 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6241 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     6301 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     6361 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagtt gggatctttg
     6421 cattggccca cggctctcag gatggggatg ctccccttca gcacccggtt ccccttggaa
     6481 actgatggtc ctggctctgt ggcatggcag tggcactgtg aggagccccc taccagcagc
     6541 acacagtggg gttggcactg ccacgctccg gatgccgcgc tctgatccaa ccccataatc
     6601 aagggaaccc gaattgcccc atcattgccc ccaccacccc catcctgccg ggccctcaca
     6661 ccccacgctg ccttctggtg acattcccca gcccaaaccc acggcttcat ggctaccgcg
     6721 gggcatttcc cattgccgcc ccattatcag ctctgcacac ctcccgctgt acccatgcct
     6781 cgtggctgcc cttctttgac gtataatctt ctaattaata cccggccttg tcaaagtgga
     6841 gcacaaacgt taattaattc cccagcaggc aggtaattaa cagtgtgact ccctttttgc
     6901 tgcgagtggg gctgatacag agagatgtgg cactatggag cccacggggt cctggcactg
     6961 ggtgcccacg gaggtcccca tgtgctgcag tgtcaccgcc tccgaggaga cagtattgtc
     7021 cctgcggtgt ccctgcagct cagctctgtc cacagggcca cctccagttt ggaggggaca
     7081 caatgcagcc ccgatgcaac ccatcctcgc agcatcccag ggacaaagac cccactgcaa
     7141 gaccgcacac agggctgggt cccgctcccc taatatctac agtgcttctt gcatggcccc
     7201 ttaatcaatg cagttaatca gcatgcgctc atgcaccgct ctggagctgc aaagcccctc
     7261 gcagcgctgc tcaccaacac cgcgcaccgc cccggcccag cctgcagcac gcgctgcaaa
     7321 caggaaagaa acaaaatatt gcccaaatgt aggcaaaggc attcggctgc cttgacctcc
     7381 gccgggccgg gccctgcctg actcagctcc ttactcagcg ctcgcttcct ccctccggct
     7441 gccaccgccg cagcgcacac cctgacaaag agtggccctt aacgggctct gaggtgcacc
     7501 cagcagtgca ctcagcagtc caagggccgg cctggaggtt tgcaccgcta cgtgctgaca
     7561 ttagcattga acttggccct gggtagtgct gcaggccggg cggggtgggt gtagagagtg
     7621 cagcgcgcgt tgcacccggt gccccttccc ctcccttgca tcccagcagg ctgcacccca
     7681 gcaccaggcc cgtgcatgca tgctcctggt gttattgcag cctggtgcat gcatgcgtct
     7741 tgatggtgca gcgctgtgca tgcatcctcc ttggtgtgta gcagtctagt gcatgcatac
     7801 ccctcggtgt tattgctgct ctgtgcacgc acgctcattg tatcacttca tcccagtgca
     7861 tgcactcaca ctggagcgat tgctgctcgg tgcacgcaca ctcattgtat cactgcagct
     7921 cagtggctgc acgcacaccg gtgttattgc tgctcggtgc gtgcatgcac atcagtgtcg
     7981 ctgcagctca gtgcatgcac gctcattgcc catcgctatc cctgcctctc ctgctggcgc
     8041 tccccgggag gtgacttcaa ggggaccgca ggaccacctc gggggtgggg ggagggctgc
     8101 acacgcggac cccgctcccc ctccccaaca aagcactgtg gaatcaaaaa ggggggaggg
     8161 gggatggagg ggcgcgtcac acccccgccc cacaccctca cctcgaggtg agccccacgt
     8221 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
     8281 tttaattatt ttgtgcagcg atgggggcgg gggggggg
//
