LOCUS       sequence-383726-        8806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rapalog-inducible coupling to human kinesin-1 (KIF5B aa1-560) via 
            FRB to anchor microtubules.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8806)
  AUTHORS   Burute M, Jansen KI, Mihajlovic M, Vermonden T, Kapitein LC
  TITLE     Local changes in microtubule network mobility instruct neuronal 
            polarization and axon specification.
  JOURNAL   Sci Adv. 2022 Nov 4;8(44):eabo2343. doi: 10.1126/sciadv.abo2343. 
            Epub 2022 Nov 4.
  PUBMED    36332030
REFERENCE   2  (bases 1 to 8806)
  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: "Sci Adv."; 
            date: "2022-11-4"; volume: "8(44):eabo2343. doi"; pages: " 
            10.1126/sciadv.abo2343. Epub 2022 Nov 4"
COMMENT     Sequence Label: pB80-hsKIF5B(1-560)-Rigor-2xmNeongreen-FRB
FEATURES             Location/Qualifiers
     source          1..8806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1024..1033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2752..3459
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      3007..3016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3487..3765
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    3964..4098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4001..4020)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4055..4074
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4112..4128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4112..4128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4125..4147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4136..4152
                     /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(4160..4166,4167..4184,4185..4190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4205..4226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4343..4360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4514..5102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4594..4613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5273..6064,6065..6133))
                     /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     5896..5915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6134..6238)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6306..6324
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6362..6384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6484..6503
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6697..6719
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6711..6728
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6712..6728
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8529..8804
                     /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 kggcggcagg kgggggkgcc gggcggggcg
      601 gggccgccyc gggccggggr gggctcgggg grggggcgcg 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 tagcgctaag
     1021 cttgccacca tgggcgcgcc tatggcggac ctggccgagt gcaacatcaa agtgatgtgt
     1081 cgcttcagac ctctcaacga gtctgaagtg aaccgcggcg acaagtacat cgccaagttt
     1141 cagggagaag acacggtcgt gatcgcgtcc aagccttatg catttgatcg ggtgttccag
     1201 tcaagcacat ctcaagagca agtgtataat gactgtgcaa agaagattgt taaagatgta
     1261 cttgaaggat ataatggaac aatatttgca tatggacaaa catcctctgg gaagacacac
     1321 acaatggagg gtaaacttca tgatccagaa ggcatgggaa ttattccaag aatagtgcaa
     1381 gatattttta attatattta ctccatggat gaaaatttgg aatttcatat taaggtttca
     1441 tattttgaaa tatatttgga taagataagg gacctgttag atgtttcaaa gaccaacctt
     1501 tcagttcatg aagacaaaaa ccgagttccc tatgtaaagg ggtgcacaga gcgttttgta
     1561 tgtagtccag atgaagttat ggataccata gatgaaggaa aatccaacag acatgtagca
     1621 gttacaaata tgaatgaaca tagctctagg agtcacagta tatttcttat taatgtcaaa
     1681 caagagaaca cacaaacgga acaaaagctg agtggaaaac tttatctggt tgatttagct
     1741 gctagtgaaa aggttagtaa aactggagct gaaggtgctg tgctggatga agctaaaaac
     1801 atcaacaagt cactttctgc tcttggaaat gttatttctg ctttggctga gggtagtaca
     1861 tatgttccat atcgagatag taaaatgaca agaatccttc aagattcatt aggtggcaac
     1921 tgtagaacca ctattgtaat ttgctgctct ccatcatcat acaatgagtc tgaaacaaaa
     1981 tctacactct tatttggcca aagggccaaa acaattaaga acacagtttg tgtcaatgtg
     2041 gagttaactg cagaacagtg gaaaaagaag tatgaaaaag aaaaagaaaa aaataagatc
     2101 ctgcggaaca ctattcagtg gcttgaaaat gagctcaaca gatggcgtaa tggggagacg
     2161 gtgcctattg atgaacagtt tgacaaagag aaagccaact tggaagcttt cacagtggat
     2221 aaagatatta ctcttaccaa tgataaacca gcaaccgcaa ttggagttat aggaaatttt
     2281 actgatgctg aaagaagaaa gtgtgaagaa gaaattgcta aattatacaa acagcttgat
     2341 gacaaggatg aagaaattaa ccagcaaagt caactggtag agaaactgaa gacgcaaatg
     2401 ttggatcagg aggagctttt ggcatctacc agaagggatc aagacaatat gcaagctgag
     2461 ctgaatcgcc ttcaagcaga aaatgatgcc tctaaagaag aagtgaaaga agttttacag
     2521 gccctagaag aacttgctgt caattatgat cagaagtctc aggaagttga agacaaaact
     2581 aaggaatatg aattgcttag tgatgaattg aatcagaaat cggcaacttt agcgagtata
     2641 gatgctgagc ttcagaaact taaggaaatg accaaccacc agaaaaaacg agcagctgag
     2701 atgatggcat ctttactaaa aaagtcgacg atccaggaag cggcaagctt aatggtgagc
     2761 aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc
     2821 tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt
     2881 tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg
     2941 gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct
     3001 ttccaggccg ccatggtaga tggctccggc taccaagtcc atcgcacaat gcagtttgaa
     3061 gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga
     3121 gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg
     3181 accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt
     3241 acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc
     3301 acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg
     3361 ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag
     3421 gcctttaccg atgtgatggg catggacgag ctgtacaagt ctagaggagg cggtggctca
     3481 actagtatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
     3541 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
     3601 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
     3661 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc
     3721 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagtaagc ggccgccata
     3781 tgcatcctag gcctattaat attccggagt atacgtagcc ggctaacgtt aacaaccggt
     3841 acctctagat aagtaatgat cataatcagc catatcacat ctgtagaggt tttacttgct
     3901 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3961 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4021 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4081 tcttatcatg tctggatcaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt
     4141 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4201 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4261 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4321 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4381 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4441 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4501 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4561 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4621 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4681 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4741 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4801 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4861 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4921 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4981 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5041 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5101 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5161 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5221 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5281 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5341 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5401 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5461 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5521 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5581 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5641 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5701 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5761 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5821 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5881 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5941 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6001 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6061 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6121 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6181 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6241 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6301 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     6361 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     6421 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     6481 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     6541 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     6601 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6661 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     6721 cggccagtgc caagttggga tctttgcatt ggcccacggc tctcaggatg gggatgctcc
     6781 ccttcagcac ccggttcccc ttggaaactg atggtcctgg ctctgtggca tggcagtggc
     6841 actgtgagga gccccctacc agcagcacac agtggggttg gcactgccac gctccggatg
     6901 ccgcgctctg atccaacccc ataatcaagg gaacccgaat tgccccatca ttgcccccac
     6961 cacccccatc ctgccgggcc ctcacacccc acgctgcctt ctggtgacat tccccagccc
     7021 aaacccacgg cttcatggct accgcggggc atttcccatt gccgccccat tatcagctct
     7081 gcacacctcc cgctgtaccc atgcctcgtg gctgcccttc tttgacgtat aatcttctaa
     7141 ttaatacccg gccttgtcaa agtggagcac aaacgttaat taattcccca gcaggcaggt
     7201 aattaacagt gtgactccct ttttgctgcg agtggggctg atacagagag atgtggcact
     7261 atggagccca cggggtcctg gcactgggtg cccacggagg tccccatgtg ctgcagtgtc
     7321 accgcctccg aggagacagt attgtccctg cggtgtccct gcagctcagc tctgtccaca
     7381 gggccacctc cagtttggag gggacacaat gcagccccga tgcaacccat cctcgcagca
     7441 tcccagggac aaagacccca ctgcaagacc gcacacaggg ctgggtcccg ctcccctaat
     7501 atctacagtg cttcttgcat ggccccttaa tcaatgcagt taatcagcat gcgctcatgc
     7561 accgctctgg agctgcaaag cccctcgcag cgctgctcac caacaccgcg caccgccccg
     7621 gcccagcctg cagcacgcgc tgcaaacagg aaagaaacaa aatattgccc aaatgtaggc
     7681 aaaggcattc ggctgccttg acctccgccg ggccgggccc tgcctgactc agctccttac
     7741 tcagcgctcg cttcctccct ccggctgcca ccgccgcagc gcacaccctg acaaagagtg
     7801 gcccttaacg ggctctgagg tgcacccagc agtgcactca gcagtccaag ggccggcctg
     7861 gaggtttgca ccgctacgtg ctgacattag cattgaactt ggccctgggt agtgctgcag
     7921 gccgggcggg gtgggtgtag agagtgcagc gcgcgttgca cccggtgccc cttcccctcc
     7981 cttgcatccc agcaggctgc accccagcac caggcccgtg catgcatgct cctggtgtta
     8041 ttgcagcctg gtgcatgcat gcgtcttgat ggtgcagcgc tgtgcatgca tcctccttgg
     8101 tgtgtagcag tctagtgcat gcatacccct cggtgttatt gctgctctgt gcacgcacgc
     8161 tcattgtatc acttcatccc agtgcatgca ctcacactgg agcgattgct gctcggtgca
     8221 cgcacactca ttgtatcact gcagctcagt ggctgcacgc acaccggtgt tattgctgct
     8281 cggtgcgtgc atgcacatca gtgtcgctgc agctcagtgc atgcacgctc attgcccatc
     8341 gctatccctg cctctcctgc tggcgctccc cgggaggtga cttcaagggg accgcaggac
     8401 cacctcgggg gtggggggag ggctgcacac gcggaccccg ctccccctcc ccaacaaagc
     8461 actgtggaat caaaaagggg ggagggggga tggaggggcg cgtcacaccc ccgccccaca
     8521 ccctcacctc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     8581 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     8641 gggggggncg cgcgccaggc ggggcggggc ggggcgrggg gcggggcggg gcgaggcgga
     8701 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     8761 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcggg
//
