LOCUS       sequence-481494-        8684 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  -end oriented kinesin for rapalog induced pulling experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8684)
  AUTHORS   Chen F, Wu J, Iwanski MK, Jurriens D, Sandron A, Pasolli M, Puma G, 
            Kromhout JZ, Yang C, Nijenhuis W, Kapitein LC, Berger F, Akhmanova A
  TITLE     Self-assembly of pericentriolar material in interphase cells lacking
            centrioles.
  JOURNAL   Elife. 2022 Jul 5;11:e77892. doi: 10.7554/eLife.77892.
  PUBMED    35787744
REFERENCE   2  (bases 1 to 8684)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-07-5"; volume: "11:e77892. doi"; pages: " 
            10.7554/eLife.77892"
COMMENT     Sequence Label: FRB-GFP-GCN4-ppKin14
FEATURES             Location/Qualifiers
     source          1..8684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             1035..1313
                     /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(1419..1421,1422..2132)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1419..1421,1422..2132)
                     /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(1461..1482)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1722..1741)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2069..2090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2142..2166
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     RBS             2630..2638
                     /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    3843..3977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3880..3899)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3934..3953
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3991..4007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3991..4007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4004..4026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4015..4031
                     /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(4039..4045,4046..4063,4064..4069))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4084..4105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4222..4239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4393..4981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4473..4492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5152..5943,5944..6012))
                     /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     5775..5794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6013..6117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6185..6203
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6241..6263)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6363..6382
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6576..6598
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6590..6607
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6591..6607
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8408..8682
                     /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 catgatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc
     1081 gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg
     1141 ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc
     1201 gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg
     1261 acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca aagcaattgt
     1321 ctagaggtgg cgggggcagt gggggagggg gaagtggagg cggcggttct ggcgggggag
     1381 gctccggtgg cggggggagc gggggtggga gtaagcttgt gagcaagggc gaggagctgt
     1441 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1501 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1561 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1621 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1681 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1741 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1801 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1861 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1921 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1981 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2041 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2101 ggatcactct cggcatggac gagctgtaca agcaccggta tacaagtttg tacaaaaaag
     2161 caggctccgc tattgaagac aagttggaag aaatactttc gaaattgtac cacattgaaa
     2221 atgagttggc cagaattaag aaattattgg gcgaaattgg tggaggtagt ggtggaggta
     2281 gtgcggccgc cagagatgat ttgaggggac gacttgtaac tctcgagaag aaattagcag
     2341 agcgtaccca agatttgagt cttgcagagt ctactttggc aactcgtgag ggtgaattcg
     2401 aatccctgca aagcaatcta atggagctta atgagctccg ggagatgaag gaggatatcg
     2461 acagaaagaa tgagcaaaca gcagctatat tgaagcgaca agcagaccag ataactgaat
     2521 tagaaactct ttatagggaa gaacaagtcc ttcgaaaacg atacttcaac atgatggaag
     2581 acatgaaagg taagatcagg gtatatgctc ggtggagacc cctaagtagt aaggaggtca
     2641 aagagcggca acaaaacgtg ttgatagcac ctgatgaatt caccatcgaa catccttgga
     2701 aagacgacaa acccaaacaa catcaatttg atcacgtttt cgaccaccat gcgacccaag
     2761 aagaggtctt tgaggataca aagtatttgg tccaatcagc aattgacgga tacaatgtct
     2821 gtatatttgc atacgggcaa actgggtcag gcaaaacgtt tacgatttac ggttctgaca
     2881 acaacccagg tctaactcca cgagctacca aagaactttt tggctatctt aagcgagacg
     2941 ccaataagtt ttcgtttgct ctgaaggttt acatgcttga aatatatcaa gattcgctca
     3001 tcgatctgct tctaccaaag agtgctgcaa agccacgaaa gcttgagatt aagaaggact
     3061 ccaagggaat ggttgtcgta gagaatgcaa ctctactgcc gattgcatcc catgatgaac
     3121 tgcaagccat tgttcataag ggtctggaga gacgacatgt ttcgggcacc cacatgaatg
     3181 cggagagttc acggtcacac ctcattctat cagtaatcgt tgaaagcacc aaccgacagt
     3241 ctcaagttct tgtgaagggc aagctgagtt ttgtggatct tgcgggctct gaaagagtga
     3301 agaaatctgg atccagtggt gaacaactga aagaggccca aagcattaat aaatctttgt
     3361 cagcactagg tgatgtcatt agcgcccttg caacagagga gcagcatatt ccttaccgta
     3421 accacaagct aacaatgctt atgagcgatt ctctcggtgg aaatgcgaag accctcatgt
     3481 ttgtaaatat ctcaccagca gagtctaatc tggacgagac ccataattca ctctgctatg
     3541 ccacaagggt aagatccatc atcaatgagg ccagtaaaaa tactacaacg aaagagatac
     3601 tgcgactgaa gaaacaaatc gcgtactgga aggagcaagc gggcaggatg ggagggtccg
     3661 aggatctgga agaggttatg gatgatcgtg gaatcaaaac tgcagattaa acgcgtaccg
     3721 gtcctagata agtaatgatc ataatcagcc atatcacatc tgtagaggtt ttacttgctt
     3781 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     3841 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3901 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3961 cttatcatgt ctggatcaat tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta
     4021 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4081 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4141 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4201 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4261 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4321 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4381 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4441 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4501 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4561 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4621 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4681 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4741 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4801 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4861 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4921 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4981 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5041 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5101 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5161 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5221 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5281 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5341 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5401 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5461 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5521 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5581 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5641 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5701 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5761 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5821 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5881 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5941 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6001 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6061 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6121 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     6181 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6241 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6301 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6361 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     6421 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     6481 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     6541 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     6601 ggccagtgcc aagttgggat ctttgcattg gcccacggct ctcaggatgg ggatgctccc
     6661 cttcagcacc cggttcccct tggaaactga tggtcctggc tctgtggcat ggcagtggca
     6721 ctgtgaggag ccccctacca gcagcacaca gtggggttgg cactgccacg ctccggatgc
     6781 cgcgctctga tccaacccca taatcaaggg aacccgaatt gccccatcat tgcccccacc
     6841 acccccatcc tgccgggccc tcacacccca cgctgccttc tggtgacatt ccccagccca
     6901 aacccacggc ttcatggcta ccgcggggca tttcccattg ccgccccatt atcagctctg
     6961 cacacctccc gctgtaccca tgcctcgtgg ctgcccttct ttgacgtata atcttctaat
     7021 taatacccgg ccttgtcaaa gtggagcaca aacgttaatt aattccccag caggcaggta
     7081 attaacagtg tgactccctt tttgctgcga gtggggctga tacagagaga tgtggcacta
     7141 tggagcccac ggggtcctgg cactgggtgc ccacggaggt ccccatgtgc tgcagtgtca
     7201 ccgcctccga ggagacagta ttgtccctgc ggtgtccctg cagctcagct ctgtccacag
     7261 ggccacctcc agtttggagg ggacacaatg cagccccgat gcaacccatc ctcgcagcat
     7321 cccagggaca aagaccccac tgcaagaccg cacacagggc tgggtcccgc tcccctaata
     7381 tctacagtgc ttcttgcatg gccccttaat caatgcagtt aatcagcatg cgctcatgca
     7441 ccgctctgga gctgcaaagc ccctcgcagc gctgctcacc aacaccgcgc accgccccgg
     7501 cccagcctgc agcacgcgct gcaaacagga aagaaacaaa atattgccca aatgtaggca
     7561 aaggcattcg gctgccttga cctccgccgg gccgggccct gcctgactca gctccttact
     7621 cagcgctcgc ttcctccctc cggctgccac cgccgcagcg cacaccctga caaagagtgg
     7681 cccttaacgg gctctgaggt gcacccagca gtgcactcag cagtccaagg gccggcctgg
     7741 aggtttgcac cgctacgtgc tgacattagc attgaacttg gccctgggta gtgctgcagg
     7801 ccgggcgggg tgggtgtaga gagtgcagcg cgcgttgcac ccggtgcccc ttcccctccc
     7861 ttgcatccca gcaggctgca ccccagcacc aggcccgtgc atgcatgctc ctggtgttat
     7921 tgcagcctgg tgcatgcatg cgtcttgatg gtgcagcgct gtgcatgcat cctccttggt
     7981 gtgtagcagt ctagtgcatg catacccctc ggtgttattg ctgctctgtg cacgcacgct
     8041 cattgtatca cttcatccca gtgcatgcac tcacactgga gcgattgctg ctcggtgcac
     8101 gcacactcat tgtatcactg cagctcagtg gctgcacgca caccggtgtt attgctgctc
     8161 ggtgcgtgca tgcacatcag tgtcgctgca gctcagtgca tgcacgctca ttgcccatcg
     8221 ctatccctgc ctctcctgct ggcgctcccc gggaggtgac ttcaagggga ccgcaggacc
     8281 acctcggggg tggggggagg gctgcacacg cggaccccgc tccccctccc caacaaagca
     8341 ctgtggaatc aaaaaggggg gaggggggat ggaggggcgc gtcacacccc cgccccacac
     8401 cctcacctcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     8461 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     8521 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga
     8581 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
     8641 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cggg
//
