LOCUS       sequence-346670-        8661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optogenetic coupling to dimeric moss kinesin-14 for retrograde 
            transport via SSPB(micro).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8661)
  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 8661)
  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-SSPB(micro)-GFP-ppKin14VIb(861-1321)
FEATURES             Location/Qualifiers
     source          1..8661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     873..895
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      958..967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(997..1203,1204..1206,1207..1326)
                     /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"
     CDS             join(1435..1437,1438..2148)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1435..1437,1438..2148)
                     /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(1477..1498)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1738..1757)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2085..2106
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             2586..2594
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    3799..3933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3836..3855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3890..3909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3947..3963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3947..3963)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3960..3982)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3971..3987
                     /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(3995..4001,4002..4019,4020..4025))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4040..4061
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4178..4195)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4349..4937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4429..4448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5108..5899,5900..5968))
                     /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     5731..5750
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5969..6073)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6141..6159
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6197..6219)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6319..6338
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6532..6554
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6546..6563
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6547..6563
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
       61 attagcgctt ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa agccttaaag
      121 ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
      181 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
      241 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
      301 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
      361 gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc acccccctcc
      421 ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtgcggggc gtggcgcggg
      481 gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc
      541 tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg
      601 aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact
      661 tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag
      721 cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg
      781 tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgtc cgcaggggga
      841 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
      901 ggtttatatc ttcccttctc tgttcctccg cagcccccaa gctaggcaat tgggcgcgcc
      961 accatggatc tgagcggcct gaccctgcag gaattcagct ccccgaaacg ccctaagctg
     1021 ctgcgtgaat attacgattg gctggttgat aacagcttta ccccatatct ggtggtggat
     1081 gccacatacc tgggcgtgaa cgtgcccgtg gagtatgtga aagacggtca gatcgtgctg
     1141 aatctgtctg caagtgcgac cggcaacctg caactgacaa atgattttat ccagttcaac
     1201 gcccagttta agggcgtgtc tcgtgaactg tatatcccga tgggtgccgc tctggccatt
     1261 tacgctcgcg agaacggcga tggtgtgatg ttcgaaccag aagaaatcta tgacgagctg
     1321 aatattggtc aattgtctag aggtggcggg ggcagtgggg gagggggaag tggaggcggc
     1381 ggttctggcg ggggaggctc cggtggcggg gggagcgggg gtgggagtaa gcttgtgagc
     1441 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1501 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1561 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1621 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1681 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1741 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1801 attgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1861 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1921 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1981 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2041 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2101 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggc tagcggaggc
     2161 ggtggctcag gcggaggagg gtcaggaggg ggaggcagtg gcgggggcgg ctctggagga
     2221 gggggttctg gtggtggtag ctacgtaaga gatgatttga ggggacgact tgtaactctc
     2281 gagaagaaat tagcagagcg tacccaagat ttgagtcttg cagagtctac tttggcaact
     2341 cgtgagggtg aattcgaatc cctgcaaagc aatctaatgg agcttaatga gctccgggag
     2401 atgaaggagg atatcgacag aaagaatgag caaacagcag ctatattgaa gcgacaagca
     2461 gaccagataa ctgaattaga aactctttat agggaagaac aagtccttcg aaaacgatac
     2521 ttcaacatga tggaagacat gaaaggtaag atcagggtat atgctcggtg gagaccccta
     2581 agtagtaagg aggtcaaaga gcggcaacaa aacgtgttga tagcacctga tgaattcacc
     2641 atcgaacatc cttggaaaga cgacaaaccc aaacaacatc aatttgatca cgttttcgac
     2701 caccatgcga cccaagaaga ggtctttgag gatacaaagt atttggtcca atcagcaatt
     2761 gacggataca atgtctgtat atttgcatac gggcaaactg ggtcaggcaa aacgtttacg
     2821 atttacggtt ctgacaacaa cccaggtcta actccacgag ctaccaaaga actttttggc
     2881 tatcttaagc gagacgccaa taagttttcg tttgctctga aggtttacat gcttgaaata
     2941 tatcaagatt cgctcatcga tctgcttcta ccaaagagtg ctgcaaagcc acgaaagctt
     3001 gagattaaga aggactccaa gggaatggtt gtcgtagaga atgcaactct actgccgatt
     3061 gcatcccatg atgaactgca agccattgtt cataagggtc tggagagacg acatgtttcg
     3121 ggcacccaca tgaatgcgga gagttcacgg tcacacctca ttctatcagt aatcgttgaa
     3181 agcaccaacc gacagtctca agttcttgtg aagggcaagc tgagttttgt ggatcttgcg
     3241 ggctctgaaa gagtgaagaa atctggatcc agtggtgaac aactgaaaga ggcccaaagc
     3301 attaataaat ctttgtcagc actaggtgat gtcattagcg cccttgcaac agaggagcag
     3361 catattcctt accgtaacca caagctaaca atgcttatga gcgattctct cggtggaaat
     3421 gcgaagaccc tcatgtttgt aaatatctca ccagcagagt ctaatctgga cgagacccat
     3481 aattcactct gctatgccac aagggtaaga tccatcatca atgaggccag taaaaatact
     3541 acaacgaaag agatactgcg actgaagaaa caaatcgcgt actggaagga gcaagcgggc
     3601 aggatgggag ggtccgagga tctggaagag gttatggatg atcgtggaat caaaactgca
     3661 gattaaacgc gtaccggtcc tagataagta atgatcataa tcagccatat cacatctgta
     3721 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3781 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3841 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3901 aaactcatca atgtatctta tcatgtctgg atcaattcgt aatcatgtca tagctgtttc
     3961 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4021 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4081 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4141 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4201 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4261 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4321 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4381 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4441 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4501 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4561 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4621 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4681 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4741 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4801 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4861 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4921 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4981 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5041 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5101 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5161 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5221 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5281 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5341 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5401 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5461 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5521 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5581 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5641 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5701 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5761 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5821 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5881 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5941 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6001 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6061 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6121 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     6181 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     6241 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     6301 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     6361 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     6421 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     6481 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     6541 gacgttgtaa aacgacggcc agtgccaagt tgggatcttt gcattggccc acggctctca
     6601 ggatggggat gctccccttc agcacccggt tccccttgga aactgatggt cctggctctg
     6661 tggcatggca gtggcactgt gaggagcccc ctaccagcag cacacagtgg ggttggcact
     6721 gccacgctcc ggatgccgcg ctctgatcca accccataat caagggaacc cgaattgccc
     6781 catcattgcc cccaccaccc ccatcctgcc gggccctcac accccacgct gccttctggt
     6841 gacattcccc agcccaaacc cacggcttca tggctaccgc ggggcatttc ccattgccgc
     6901 cccattatca gctctgcaca cctcccgctg tacccatgcc tcgtggctgc ccttctttga
     6961 cgtataatct tctaattaat acccggcctt gtcaaagtgg agcacaaacg ttaattaatt
     7021 ccccagcagg caggtaatta acagtgtgac tccctttttg ctgcgagtgg ggctgataca
     7081 gagagatgtg gcactatgga gcccacgggg tcctggcact gggtgcccac ggaggtcccc
     7141 atgtgctgca gtgtcaccgc ctccgaggag acagtattgt ccctgcggtg tccctgcagc
     7201 tcagctctgt ccacagggcc acctccagtt tggaggggac acaatgcagc cccgatgcaa
     7261 cccatcctcg cagcatccca gggacaaaga ccccactgca agaccgcaca cagggctggg
     7321 tcccgctccc ctaatatcta cagtgcttct tgcatggccc cttaatcaat gcagttaatc
     7381 agcatgcgct catgcaccgc tctggagctg caaagcccct cgcagcgctg ctcaccaaca
     7441 ccgcgcaccg ccccggccca gcctgcagca cgcgctgcaa acaggaaaga aacaaaatat
     7501 tgcccaaatg taggcaaagg cattcggctg ccttgacctc cgccgggccg ggccctgcct
     7561 gactcagctc cttactcagc gctcgcttcc tccctccggc tgccaccgcc gcagcgcaca
     7621 ccctgacaaa gagtggccct taacgggctc tgaggtgcac ccagcagtgc actcagcagt
     7681 ccaagggccg gcctggaggt ttgcaccgct acgtgctgac attagcattg aacttggccc
     7741 tgggtagtgc tgcaggccgg gcggggtggg tgtagagagt gcagcgcgcg ttgcacccgg
     7801 tgccccttcc cctcccttgc atcccagcag gctgcacccc agcaccaggc ccgtgcatgc
     7861 atgctcctgg tgttattgca gcctggtgca tgcatgcgtc ttgatggtgc agcgctgtgc
     7921 atgcatcctc cttggtgtgt agcagtctag tgcatgcata cccctcggtg ttattgctgc
     7981 tctgtgcacg cacgctcatt gtatcacttc atcccagtgc atgcactcac actggagcga
     8041 ttgctgctcg gtgcacgcac actcattgta tcactgcagc tcagtggctg cacgcacacc
     8101 ggtgttattg ctgctcggtg cgtgcatgca catcagtgtc gctgcagctc agtgcatgca
     8161 cgctcattgc ccatcgctat ccctgcctct cctgctggcg ctccccggga ggtgacttca
     8221 aggggaccgc aggaccacct cgggggtggg gggagggctg cacacgcgga ccccgctccc
     8281 cctccccaac aaagcactgt ggaatcaaaa aggggggagg ggggatggag gggcgcgtca
     8341 cacccccgcc ccacaccctc acctcgaggt gagccccacg ttctgcttca ctctccccat
     8401 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     8461 gatgggggcg ggggggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
     8521 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
     8581 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
     8641 cgcgccgccc gccccggctc t
//
