LOCUS       sequence-346989-        8725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Poorly reversible opto-kinesin for optogenetic heterodimerization to
            iLID via SSPB(micro).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8725)
  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 8725)
  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)-VVD52C-mCherry-SSPB(micro)
FEATURES             Location/Qualifiers
     source          1..8725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1059..1081
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1144..1153
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2126..2135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2749..3453
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2891..2909)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             join(3583..3789,3790..3792,3793..3912)
                     /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    4045..4179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4082..4101)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4136..4155
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4193..4209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4193..4209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4206..4228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4217..4233
                     /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(4241..4247,4248..4265,4266..4271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4286..4307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4424..4441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4595..5183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4675..4694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5354..6145,6146..6214))
                     /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     5977..5996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6215..6319)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6387..6405
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6443..6465)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6565..6584
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6778..6800
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6792..6809
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6793..6809
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
       61 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      121 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      181 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      241 gctgtaatta gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc
      301 ttaaagggct ccgggagggc cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg
      361 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      421 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
      481 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
      541 gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc
      601 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg
      661 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg
      721 gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg
      781 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
      841 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc
      901 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
      961 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgtccgca
     1021 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
     1081 cggcggggtt tatatcttcc cttctctgtt cctccgcagc ccccaagcta ggcaattggg
     1141 cgcgccacca tggccggggc ttcggtgaag gtggcggtgc gtgtccgccc cttcaactcc
     1201 cgggaaatga gccgagactc caagtgcatc attcagatgt ctggaagcac caccactatt
     1261 gtcaacccca aacagcccaa ggagacaccc aagagcttca gctttgatta ctcctactgg
     1321 tcacatacct cgccggagga catcaactat gcctctcaga agcaggtcta cagggacatt
     1381 ggggaagaga tgctacagca tgcctttgag ggctacaatg tgtgcatctt tgcctatggg
     1441 cagacggggg ccggcaagtc ctacaccatg atggggaaac aggagaagga ccagcaaggc
     1501 atcattccac agctctgcga agacctcttc tctcgaatca acgacaccac caacgacaac
     1561 atgtcctact ctgtggaggt cagctacatg gagatttact gtgagcgagt ccgtgacctc
     1621 ctgaacccca agaacaaagg caacctgcgt gtgagggagc accctctgct ggggccttat
     1681 gtggaggacc tctccaagct ggctgtaacc tcctacaatg acatccagga cctcatggac
     1741 tcagggaaca aggccaggac tgtggcggcc acaaacatga atgagaccag cagccgttct
     1801 cacgcagtct tcaacatcat ctttacccag aagcgccatg atgcagagac caacatcacc
     1861 acagagaaag tgagcaaaat cagcctggtg gacctggcag gcagtgaacg agccgactcc
     1921 acaggagcca agggcacgcg actcaaggag ggggccaaca tcaacaagtc tttgaccacc
     1981 ctgggaaagg tcatctctgc tctggctgaa atggactctg gacccaacaa gaacaagaaa
     2041 aagaagaaga cagatttcat cccttaccga gactccgtat tgacatggct tctccgggaa
     2101 aacctcggtg gcaactcaag gacagccatg gtggcagccc tgagccctgc ggacatcaac
     2161 tatgatgaga ccctcagcac actgagatat gcagatcggg ccaaacaaat ccgctgcaat
     2221 gccatcatca atgaagaccc caactctaga ggtggcgggg gcagtggggg atcccatact
     2281 ctttatgccc ccggtggata tgacattatg ggatatctgt gccagatcat gaaccggcca
     2341 aacccgcagg tggaactggg ccccgtggat acatcctgcg ccttgattct ttgtgacctg
     2401 aaacagaaag acaccccgat agtttacgcg agtgaagcct tcctctacat gacaggttac
     2461 agcaacgcag aggtgctggg ccggaattgc cggtttctgc aaagccctga cggcatggtg
     2521 aagcccaaga gcacccggaa gtacgtggat agtaacacaa tcaatactat gcgcaaggca
     2581 atcgacagga atgccgaggt gcaggttgaa gtagtcaatt ttaaaaagaa tggacagcga
     2641 tttgttaatt tcctgactat gatacctgtt agggacgaaa caggcgagta tcgatactct
     2701 atgggattcc agtgcgaagt cgacgggagc gggggtggga gtaagcttgt gagcaagggc
     2761 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     2821 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2881 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2941 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     3001 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     3061 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     3121 aaggtgaagc tgcgcggcac caacttcccc tccgacggtc ccgtaatgca gaagaagacc
     3181 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3241 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     3301 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     3361 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     3421 cactccaccg gcggcatgga cgagctgtac aaggctagcg gaggcggtgg ctcaggcgga
     3481 ggagggtcag gagggggagg cagtggcggg ggcggctctg gaggaggggg ttctggtggt
     3541 ggtagctacg tagatctgag cggcctgacc ctgcaggaat tcagctcccc gaaacgccct
     3601 aagctgctgc gtgaatatta cgattggctg gttgataaca gctttacccc atatctggtg
     3661 gtggatgcca catacctggg cgtgaacgtg cccgtggagt atgtgaaaga cggtcagatc
     3721 gtgctgaatc tgtctgcaag tgcgaccggc aacctgcaac tgacaaatga ttttatccag
     3781 ttcaacgccc agtttaaggg cgtgtctcgt gaactgtata tcccgatggg tgccgctctg
     3841 gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg aaccagaaga aatctatgac
     3901 gagctgaata ttggttaaac cggtcctaga taagtaatga tcataatcag ccatatcaca
     3961 tctgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     4021 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     4081 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4141 ttgtccaaac tcatcaatgt atcttatcat gtctggatca attcgtaatc atgtcatagc
     4201 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4261 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4321 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4381 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4441 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4501 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4561 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4621 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4681 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4741 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4801 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4861 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4921 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4981 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5041 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5101 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5161 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5221 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5281 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5341 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5401 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5461 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5521 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5581 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5641 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5701 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5761 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5821 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5881 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5941 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6001 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6061 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6121 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6181 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6241 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6301 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6361 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6421 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6481 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6541 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     6601 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
     6661 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     6721 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     6781 agtcacgacg ttgtaaaacg acggccagtg ccaagttggg atctttgcat tggcccacgg
     6841 ctctcaggat ggggatgctc cccttcagca cccggttccc cttggaaact gatggtcctg
     6901 gctctgtggc atggcagtgg cactgtgagg agccccctac cagcagcaca cagtggggtt
     6961 ggcactgcca cgctccggat gccgcgctct gatccaaccc cataatcaag ggaacccgaa
     7021 ttgccccatc attgccccca ccacccccat cctgccgggc cctcacaccc cacgctgcct
     7081 tctggtgaca ttccccagcc caaacccacg gcttcatggc taccgcgggg catttcccat
     7141 tgccgcccca ttatcagctc tgcacacctc ccgctgtacc catgcctcgt ggctgccctt
     7201 ctttgacgta taatcttcta attaataccc ggccttgtca aagtggagca caaacgttaa
     7261 ttaattcccc agcaggcagg taattaacag tgtgactccc tttttgctgc gagtggggct
     7321 gatacagaga gatgtggcac tatggagccc acggggtcct ggcactgggt gcccacggag
     7381 gtccccatgt gctgcagtgt caccgcctcc gaggagacag tattgtccct gcggtgtccc
     7441 tgcagctcag ctctgtccac agggccacct ccagtttgga ggggacacaa tgcagccccg
     7501 atgcaaccca tcctcgcagc atcccaggga caaagacccc actgcaagac cgcacacagg
     7561 gctgggtccc gctcccctaa tatctacagt gcttcttgca tggcccctta atcaatgcag
     7621 ttaatcagca tgcgctcatg caccgctctg gagctgcaaa gcccctcgca gcgctgctca
     7681 ccaacaccgc gcaccgcccc ggcccagcct gcagcacgcg ctgcaaacag gaaagaaaca
     7741 aaatattgcc caaatgtagg caaaggcatt cggctgcctt gacctccgcc gggccgggcc
     7801 ctgcctgact cagctcctta ctcagcgctc gcttcctccc tccggctgcc accgccgcag
     7861 cgcacaccct gacaaagagt ggcccttaac gggctctgag gtgcacccag cagtgcactc
     7921 agcagtccaa gggccggcct ggaggtttgc accgctacgt gctgacatta gcattgaact
     7981 tggccctggg tagtgctgca ggccgggcgg ggtgggtgta gagagtgcag cgcgcgttgc
     8041 acccggtgcc ccttcccctc ccttgcatcc cagcaggctg caccccagca ccaggcccgt
     8101 gcatgcatgc tcctggtgtt attgcagcct ggtgcatgca tgcgtcttga tggtgcagcg
     8161 ctgtgcatgc atcctccttg gtgtgtagca gtctagtgca tgcatacccc tcggtgttat
     8221 tgctgctctg tgcacgcacg ctcattgtat cacttcatcc cagtgcatgc actcacactg
     8281 gagcgattgc tgctcggtgc acgcacactc attgtatcac tgcagctcag tggctgcacg
     8341 cacaccggtg ttattgctgc tcggtgcgtg catgcacatc agtgtcgctg cagctcagtg
     8401 catgcacgct cattgcccat cgctatccct gcctctcctg ctggcgctcc ccgggaggtg
     8461 acttcaaggg gaccgcagga ccacctcggg ggtgggggga gggctgcaca cgcggacccc
     8521 gctccccctc cccaacaaag cactgtggaa tcaaaaaggg gggagggggg atggaggggc
     8581 gcgtcacacc cccgccccac accctcacct cgaggtgagc cccacgttct gcttcactct
     8641 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     8701 tgcagcgatg ggggcggggg ggggg
//
