LOCUS       sequence-345348-        8433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively active KIF1A for optogenetic heterodimerization to 
            iLID via SSPB(milli).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8433)
  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 8433)
  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(milli)
FEATURES             Location/Qualifiers
     source          1..8433
                     /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
                     IVLNLSASVTGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    3591..3725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3628..3647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3682..3701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3739..3755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3739..3755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3752..3774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3763..3779
                     /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(3787..3793,3794..3811,3812..3817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3832..3853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3970..3987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4141..4729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4221..4240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4900..5691,5692..5760))
                     /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     5523..5542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5761..5865)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5933..5951
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5989..6011)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6111..6130
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6324..6346
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6338..6355
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6339..6355
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8156..8431
                     /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 ggctccggtg gcggggggag cgggggtggg 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 tgtgaccggc aacctgcaac
     3301 tgacaaatga ttttatccag ttcaacgccc agtttaaggg cgtgtctcgt gaactgtata
     3361 tcccgatggg tgccgctctg gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg
     3421 aaccagaaga aatctatgac gagctgaata ttggttaaac gcgtaccggt cctagataag
     3481 taatgatcat aatcagccat atcacatctg tagaggtttt acttgcttta aaaaacctcc
     3541 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     3601 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3661 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3721 ggatcaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3781 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3841 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3961 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4021 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4741 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4801 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4981 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5461 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5761 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5821 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5881 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5941 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     6001 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     6061 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     6121 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     6181 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     6241 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     6301 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
     6361 gttgggatct ttgcattggc ccacggctct caggatgggg atgctcccct tcagcacccg
     6421 gttccccttg gaaactgatg gtcctggctc tgtggcatgg cagtggcact gtgaggagcc
     6481 ccctaccagc agcacacagt ggggttggca ctgccacgct ccggatgccg cgctctgatc
     6541 caaccccata atcaagggaa cccgaattgc cccatcattg cccccaccac ccccatcctg
     6601 ccgggccctc acaccccacg ctgccttctg gtgacattcc ccagcccaaa cccacggctt
     6661 catggctacc gcggggcatt tcccattgcc gccccattat cagctctgca cacctcccgc
     6721 tgtacccatg cctcgtggct gcccttcttt gacgtataat cttctaatta atacccggcc
     6781 ttgtcaaagt ggagcacaaa cgttaattaa ttccccagca ggcaggtaat taacagtgtg
     6841 actccctttt tgctgcgagt ggggctgata cagagagatg tggcactatg gagcccacgg
     6901 ggtcctggca ctgggtgccc acggaggtcc ccatgtgctg cagtgtcacc gcctccgagg
     6961 agacagtatt gtccctgcgg tgtccctgca gctcagctct gtccacaggg ccacctccag
     7021 tttggagggg acacaatgca gccccgatgc aacccatcct cgcagcatcc cagggacaaa
     7081 gaccccactg caagaccgca cacagggctg ggtcccgctc ccctaatatc tacagtgctt
     7141 cttgcatggc cccttaatca atgcagttaa tcagcatgcg ctcatgcacc gctctggagc
     7201 tgcaaagccc ctcgcagcgc tgctcaccaa caccgcgcac cgccccggcc cagcctgcag
     7261 cacgcgctgc aaacaggaaa gaaacaaaat attgcccaaa tgtaggcaaa ggcattcggc
     7321 tgccttgacc tccgccgggc cgggccctgc ctgactcagc tccttactca gcgctcgctt
     7381 cctccctccg gctgccaccg ccgcagcgca caccctgaca aagagtggcc cttaacgggc
     7441 tctgaggtgc acccagcagt gcactcagca gtccaagggc cggcctggag gtttgcaccg
     7501 ctacgtgctg acattagcat tgaacttggc cctgggtagt gctgcaggcc gggcggggtg
     7561 ggtgtagaga gtgcagcgcg cgttgcaccc ggtgcccctt cccctccctt gcatcccagc
     7621 aggctgcacc ccagcaccag gcccgtgcat gcatgctcct ggtgttattg cagcctggtg
     7681 catgcatgcg tcttgatggt gcagcgctgt gcatgcatcc tccttggtgt gtagcagtct
     7741 agtgcatgca tacccctcgg tgttattgct gctctgtgca cgcacgctca ttgtatcact
     7801 tcatcccagt gcatgcactc acactggagc gattgctgct cggtgcacgc acactcattg
     7861 tatcactgca gctcagtggc tgcacgcaca ccggtgttat tgctgctcgg tgcgtgcatg
     7921 cacatcagtg tcgctgcagc tcagtgcatg cacgctcatt gcccatcgct atccctgcct
     7981 ctcctgctgg cgctccccgg gaggtgactt caaggggacc gcaggaccac ctcgggggtg
     8041 gggggagggc tgcacacgcg gaccccgctc cccctcccca acaaagcact gtggaatcaa
     8101 aaagggggga ggggggatgg aggggcgcgt cacacccccg ccccacaccc tcacctcgag
     8161 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     8221 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggcgcgc
     8281 gccaggcggg gcggggsggg gsgrggggsg gggcggggcg aggcggagag gtgcggcggc
     8341 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     8401 gccctataaa aagcgaagcg cgcggcgggc ggg
//
