LOCUS       sequence-346971-        8427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively active 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 8427)
  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 8427)
  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(1-383)-GFP-SSPB(micro)
FEATURES             Location/Qualifiers
     source          1..8427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1026..1035
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2008..2017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2280..2282,2283..2993)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2280..2282,2283..2993)
                     /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(2322..2343)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2583..2602)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2930..2951
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(3123..3329,3330..3332,3333..3452)
                     /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    3585..3719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3622..3641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3676..3695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3733..3749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3733..3749)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3746..3768)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3757..3773
                     /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(3781..3787,3788..3805,3806..3811))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3826..3847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3964..3981)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4135..4723)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4215..4234)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4894..5685,5686..5754))
                     /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     5517..5536
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5755..5859)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5927..5945
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5983..6005)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6105..6124
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6318..6340
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6332..6349
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6333..6349
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8150..8425
                     /label=chicken beta-actin promoter
ORIGIN
        1 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
       61 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      121 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      181 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      241 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      301 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
      361 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
      421 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
      481 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tgcggggcgt
      541 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
      601 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
      661 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
      721 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
      781 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
      841 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc tccagcctcg gggctgtccg
      901 cagggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
      961 accggcgggg tttatatctt cccttctctg ttcctccgca gcccccaagc taggcaattg
     1021 ggcgcgccac catggccggg gcttcggtga aggtggcggt gcgtgtccgc cccttcaact
     1081 cccgggaaat gagccgagac tccaagtgca tcattcagat gtctggaagc accaccacta
     1141 ttgtcaaccc caaacagccc aaggagacac ccaagagctt cagctttgat tactcctact
     1201 ggtcacatac ctcgccggag gacatcaact atgcctctca gaagcaggtc tacagggaca
     1261 ttggggaaga gatgctacag catgcctttg agggctacaa tgtgtgcatc tttgcctatg
     1321 ggcagacggg ggccggcaag tcctacacca tgatggggaa acaggagaag gaccagcaag
     1381 gcatcattcc acagctctgc gaagacctct tctctcgaat caacgacacc accaacgaca
     1441 acatgtccta ctctgtggag gtcagctaca tggagattta ctgtgagcga gtccgtgacc
     1501 tcctgaaccc caagaacaaa ggcaacctgc gtgtgaggga gcaccctctg ctggggcctt
     1561 atgtggagga cctctccaag ctggctgtaa cctcctacaa tgacatccag gacctcatgg
     1621 actcagggaa caaggccagg actgtggcgg ccacaaacat gaatgagacc agcagccgtt
     1681 ctcacgcagt cttcaacatc atctttaccc agaagcgcca tgatgcagag accaacatca
     1741 ccacagagaa agtgagcaaa atcagcctgg tggacctggc aggcagtgaa cgagccgact
     1801 ccacaggagc caagggcacg cgactcaagg agggggccaa catcaacaag tctttgacca
     1861 ccctgggaaa ggtcatctct gctctggctg aaatggactc tggacccaac aagaacaaga
     1921 aaaagaagaa gacagatttc atcccttacc gagactccgt attgacatgg cttctccggg
     1981 aaaacctcgg tggcaactca aggacagcca tggtggcagc cctgagccct gcggacatca
     2041 actatgatga gaccctcagc acactgagat atgcagatcg ggccaaacaa atccgctgca
     2101 atgccatcat caatgaagac cccaacaaca agctgattag agaactgaag gatgaggtga
     2161 cccggctgcg ggaccttctg tctagaggtg gcgggggcag tgggggaggg ggaagtggag
     2221 gcggcggttc tggcggggga ggctccggkg gsggggggcg sgggggkggg agtaagcttg
     2281 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2341 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2401 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2461 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2521 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2581 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2641 accgcattga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2701 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2761 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2821 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2881 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2941 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggctagcg
     3001 gaggcggtgg ctcaggcgga ggagggtcag gagggggagg cagtggcggg ggcggctctg
     3061 gaggaggggg ttctggtggt ggtagctacg tagatctgag cggcctgacc ctgcaggaat
     3121 tcagctcccc gaaacgccct aagctgctgc gtgaatatta cgattggctg gttgataaca
     3181 gctttacccc atatctggtg gtggatgcca catacctggg cgtgaacgtg cccgtggagt
     3241 atgtgaaaga cggtcagatc gtgctgaatc tgtctgcaag tgcgaccggc aacctgcaac
     3301 tgacaaatga ttttatccag ttcaacgccc agtttaaggg cgtgtctcgt gaactgtata
     3361 tcccgatggg tgccgctctg gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg
     3421 aaccagaaga aatctatgac gagctgaata ttggttaaac cggtcctaga taagtaatga
     3481 tcataatcag ccatatcaca tctgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3541 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3601 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3661 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca
     3721 attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3781 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3841 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3901 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3961 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4021 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4081 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4141 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4201 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4261 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4321 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4381 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4441 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4501 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4561 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4621 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4681 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4741 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4801 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4861 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4921 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4981 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5041 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5101 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5161 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5221 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5281 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5341 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5401 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5461 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5521 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5581 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5641 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5701 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5761 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5821 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5881 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     5941 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     6001 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     6061 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     6121 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     6181 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6241 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     6301 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagttggg
     6361 atctttgcat tggcccacgg ctctcaggat ggggatgctc cccttcagca cccggttccc
     6421 cttggaaact gatggtcctg gctctgtggc atggcagtgg cactgtgagg agccccctac
     6481 cagcagcaca cagtggggtt ggcactgcca cgctccggat gccgcgctct gatccaaccc
     6541 cataatcaag ggaacccgaa ttgccccatc attgccccca ccacccccat cctgccgggc
     6601 cctcacaccc cacgctgcct tctggtgaca ttccccagcc caaacccacg gcttcatggc
     6661 taccgcgggg catttcccat tgccgcccca ttatcagctc tgcacacctc ccgctgtacc
     6721 catgcctcgt ggctgccctt ctttgacgta taatcttcta attaataccc ggccttgtca
     6781 aagtggagca caaacgttaa ttaattcccc agcaggcagg taattaacag tgtgactccc
     6841 tttttgctgc gagtggggct gatacagaga gatgtggcac tatggagccc acggggtcct
     6901 ggcactgggt gcccacggag gtccccatgt gctgcagtgt caccgcctcc gaggagacag
     6961 tattgtccct gcggtgtccc tgcagctcag ctctgtccac agggccacct ccagtttgga
     7021 ggggacacaa tgcagccccg atgcaaccca tcctcgcagc atcccaggga caaagacccc
     7081 actgcaagac cgcacacagg gctgggtccc gctcccctaa tatctacagt gcttcttgca
     7141 tggcccctta atcaatgcag ttaatcagca tgcgctcatg caccgctctg gagctgcaaa
     7201 gcccctcgca gcgctgctca ccaacaccgc gcaccgcccc ggcccagcct gcagcacgcg
     7261 ctgcaaacag gaaagaaaca aaatattgcc caaatgtagg caaaggcatt cggctgcctt
     7321 gacctccgcc gggccgggcc ctgcctgact cagctcctta ctcagcgctc gcttcctccc
     7381 tccggctgcc accgccgcag cgcacaccct gacaaagagt ggcccttaac gggctctgag
     7441 gtgcacccag cagtgcactc agcagtccaa gggccggcct ggaggtttgc accgctacgt
     7501 gctgacatta gcattgaact tggccctggg tagtgctgca ggccgggcgg ggtgggtgta
     7561 gagagtgcag cgcgcgttgc acccggtgcc ccttcccctc ccttgcatcc cagcaggctg
     7621 caccccagca ccaggcccgt gcatgcatgc tcctggtgtt attgcagcct ggtgcatgca
     7681 tgcgtcttga tggtgcagcg ctgtgcatgc atcctccttg gtgtgtagca gtctagtgca
     7741 tgcatacccc tcggtgttat tgctgctctg tgcacgcacg ctcattgtat cacttcatcc
     7801 cagtgcatgc actcacactg gagcgattgc tgctcggtgc acgcacactc attgtatcac
     7861 tgcagctcag tggctgcacg cacaccggtg ttattgctgc tcggtgcgtg catgcacatc
     7921 agtgtcgctg cagctcagtg catgcacgct cattgcccat cgctatccct gcctctcctg
     7981 ctggcgctcc ccgggaggtg acttcaaggg gaccgcagga ccacctcggg ggtgggggga
     8041 gggctgcaca cgcggacccc gctccccctc cccaacaaag cactgtggaa tcaaaaaggg
     8101 gggagggggg atggaggggc gcgtcacacc cccgccccac accctcacct cgaggtgagc
     8161 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
     8221 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggc gcgcgccagg
     8281 cggggcgggg cggggcgrgg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
     8341 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
     8401 taaaaagcga agcgcgcggc gggcggg
//
