LOCUS       sequence-346987-        8567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rapalog-inducible coupling to dimeric moss kinesin-14 for retrograde
            transport via FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8567)
  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 8567)
  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-FRB-GFP-ppKin14VIb(861-1321)
FEATURES             Location/Qualifiers
     source          1..8567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1053..1075
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             1147..1425
                     /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"
     CDS             join(1531..1533,1534..2244)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1531..1533,1534..2244)
                     /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(1573..1594)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1834..1853)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2181..2202
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             2682..2690
                     /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    3895..4029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3932..3951)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3986..4005
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4043..4059)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4043..4059)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4056..4078)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4067..4083
                     /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(4091..4097,4098..4115,4116..4121))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4136..4157
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4274..4291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4445..5033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4525..4544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5204..5995,5996..6064))
                     /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     5827..5846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6065..6169)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6237..6255
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6293..6315)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6415..6434
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6628..6650
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6642..6659
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6643..6659
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
       61 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
      121 cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct
      181 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
      241 attagcgctt ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa agccttaaag
      301 ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
      361 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
      421 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
      481 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
      541 gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc acccccctcc
      601 ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtgcggggc gtggcgcggg
      661 gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc
      721 tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg
      781 aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact
      841 tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag
      901 cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg
      961 tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgtc cgcaggggga
     1021 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     1081 ggtttatatc ttcccttctc tgttcctccg cagcccccaa gctaggcaat tgggcgcgcc
     1141 accatgatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
     1201 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
     1261 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
     1321 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc
     1381 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagcaatt gtctagaggt
     1441 ggcgggggca gtgggggagg gggaagtgga ggcggcggtt ctggcggggg aggctccggt
     1501 ggcgggggga gcgggggtgg gagtaagctt gtgagcaagg gcgaggagct gttcaccggg
     1561 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1621 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1681 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1741 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1801 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1861 gaggtgaagt tcgagggcga caccctggtg aaccgcattg agctgaaggg catcgacttc
     1921 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1981 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2041 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2101 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2161 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2221 ctcggcatgg acgagctgta caaggctagc ggaggcggtg gctcaggcgg aggagggtca
     2281 ggagggggag gcagtggcgg gggcggctct ggaggagggg gttctggtgg tggtagctac
     2341 gtaagagatg atttgagggg acgacttgta actctcgaga agaaattagc agagcgtacc
     2401 caagatttga gtcttgcaga gtctactttg gcaactcgtg agggtgaatt cgaatccctg
     2461 caaagcaatc taatggagct taatgagctc cgggagatga aggaggatat cgacagaaag
     2521 aatgagcaaa cagcagctat attgaagcga caagcagacc agataactga attagaaact
     2581 ctttataggg aagaacaagt ccttcgaaaa cgatacttca acatgatgga agacatgaaa
     2641 ggtaagatca gggtatatgc tcggtggaga cccctaagta gtaaggaggt caaagagcgg
     2701 caacaaaacg tgttgatagc acctgatgaa ttcaccatcg aacatccttg gaaagacgac
     2761 aaacccaaac aacatcaatt tgatcacgtt ttcgaccacc atgcgaccca agaagaggtc
     2821 tttgaggata caaagtattt ggtccaatca gcaattgacg gatacaatgt ctgtatattt
     2881 gcatacgggc aaactgggtc aggcaaaacg tttacgattt acggttctga caacaaccca
     2941 ggtctaactc cacgagctac caaagaactt tttggctatc ttaagcgaga cgccaataag
     3001 ttttcgtttg ctctgaaggt ttacatgctt gaaatatatc aagattcgct catcgatctg
     3061 cttctaccaa agagtgctgc aaagccacga aagcttgaga ttaagaagga ctccaaggga
     3121 atggttgtcg tagagaatgc aactctactg ccgattgcat cccatgatga actgcaagcc
     3181 attgttcata agggtctgga gagacgacat gtttcgggca cccacatgaa tgcggagagt
     3241 tcacggtcac acctcattct atcagtaatc gttgaaagca ccaaccgaca gtctcaagtt
     3301 cttgtgaagg gcaagctgag ttttgtggat cttgcgggct ctgaaagagt gaagaaatct
     3361 ggatccagtg gtgaacaact gaaagaggcc caaagcatta ataaatcttt gtcagcacta
     3421 ggtgatgtca ttagcgccct tgcaacagag gagcagcata ttccttaccg taaccacaag
     3481 ctaacaatgc ttatgagcga ttctctcggt ggaaatgcga agaccctcat gtttgtaaat
     3541 atctcaccag cagagtctaa tctggacgag acccataatt cactctgcta tgccacaagg
     3601 gtaagatcca tcatcaatga ggccagtaaa aatactacaa cgaaagagat actgcgactg
     3661 aagaaacaaa tcgcgtactg gaaggagcaa gcgggcagga tgggagggtc cgaggatctg
     3721 gaagaggtta tggatgatcg tggaatcaaa actgcagatt aaacgcgtac cggtcctaga
     3781 taagtaatga tcataatcag ccatatcaca tctgtagagg ttttacttgc tttaaaaaac
     3841 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3901 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3961 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4021 gtctggatca attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4081 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4141 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4201 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4261 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4321 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4381 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4441 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4501 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4561 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4621 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4681 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4741 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4801 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4861 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4921 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4981 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5041 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5101 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5161 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5221 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5281 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5341 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5401 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5461 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5521 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5581 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5641 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5701 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5761 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5821 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5881 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5941 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6001 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6061 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6121 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6181 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6241 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6301 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6361 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6421 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     6481 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     6541 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6601 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     6661 ccaagttggg atctttgcat tggcccacgg ctctcaggat ggggatgctc cccttcagca
     6721 cccggttccc cttggaaact gatggtcctg gctctgtggc atggcagtgg cactgtgagg
     6781 agccccctac cagcagcaca cagtggggtt ggcactgcca cgctccggat gccgcgctct
     6841 gatccaaccc cataatcaag ggaacccgaa ttgccccatc attgccccca ccacccccat
     6901 cctgccgggc cctcacaccc cacgctgcct tctggtgaca ttccccagcc caaacccacg
     6961 gcttcatggc taccgcgggg catttcccat tgccgcccca ttatcagctc tgcacacctc
     7021 ccgctgtacc catgcctcgt ggctgccctt ctttgacgta taatcttcta attaataccc
     7081 ggccttgtca aagtggagca caaacgttaa ttaattcccc agcaggcagg taattaacag
     7141 tgtgactccc tttttgctgc gagtggggct gatacagaga gatgtggcac tatggagccc
     7201 acggggtcct ggcactgggt gcccacggag gtccccatgt gctgcagtgt caccgcctcc
     7261 gaggagacag tattgtccct gcggtgtccc tgcagctcag ctctgtccac agggccacct
     7321 ccagtttgga ggggacacaa tgcagccccg atgcaaccca tcctcgcagc atcccaggga
     7381 caaagacccc actgcaagac cgcacacagg gctgggtccc gctcccctaa tatctacagt
     7441 gcttcttgca tggcccctta atcaatgcag ttaatcagca tgcgctcatg caccgctctg
     7501 gagctgcaaa gcccctcgca gcgctgctca ccaacaccgc gcaccgcccc ggcccagcct
     7561 gcagcacgcg ctgcaaacag gaaagaaaca aaatattgcc caaatgtagg caaaggcatt
     7621 cggctgcctt gacctccgcc gggccgggcc ctgcctgact cagctcctta ctcagcgctc
     7681 gcttcctccc tccggctgcc accgccgcag cgcacaccct gacaaagagt ggcccttaac
     7741 gggctctgag gtgcacccag cagtgcactc agcagtccaa gggccggcct ggaggtttgc
     7801 accgctacgt gctgacatta gcattgaact tggccctggg tagtgctgca ggccgggcgg
     7861 ggtgggtgta gagagtgcag cgcgcgttgc acccggtgcc ccttcccctc ccttgcatcc
     7921 cagcaggctg caccccagca ccaggcccgt gcatgcatgc tcctggtgtt attgcagcct
     7981 ggtgcatgca tgcgtcttga tggtgcagcg ctgtgcatgc atcctccttg gtgtgtagca
     8041 gtctagtgca tgcatacccc tcggtgttat tgctgctctg tgcacgcacg ctcattgtat
     8101 cacttcatcc cagtgcatgc actcacactg gagcgattgc tgctcggtgc acgcacactc
     8161 attgtatcac tgcagctcag tggctgcacg cacaccggtg ttattgctgc tcggtgcgtg
     8221 catgcacatc agtgtcgctg cagctcagtg catgcacgct cattgcccat cgctatccct
     8281 gcctctcctg ctggcgctcc ccgggaggtg acttcaaggg gaccgcagga ccacctcggg
     8341 ggtgggggga gggctgcaca cgcggacccc gctccccctc cccaacaaag cactgtggaa
     8401 tcaaaaaggg gggagggggg atggaggggc gcgtcacacc cccgccccac accctcacct
     8461 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     8521 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcgg
//
