LOCUS       sequence-481493-        8594 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 8594)
  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 8594)
  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-BFP-GCN4-ppKin14
FEATURES             Location/Qualifiers
     source          1..8594
                     /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             1362..2057
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     RBS             2540..2548
                     /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    3753..3887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3790..3809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3844..3863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3901..3917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3901..3917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3914..3936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3925..3941
                     /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(3949..3955,3956..3973,3974..3979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3994..4015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4132..4149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4303..4891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4383..4402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5062..5853,5854..5922))
                     /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     5685..5704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5923..6027)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6095..6113
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6151..6173)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6273..6292
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6486..6508
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6500..6517
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6501..6517
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8318..8592
                     /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 cgacaggatc cgctggctcc gctgctggtt ctggcgaatt cagcgagctg attaaggaga
     1381 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     1441 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     1501 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     1561 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     1621 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     1681 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     1741 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     1801 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     1861 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     1921 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     1981 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     2041 tggggcacaa gcttaatgtg tacaaaaaag caggctccgc tattgaagac aagttggaag
     2101 aaatactttc gaaattgtac cacattgaaa atgagttggc cagaattaag aaattattgg
     2161 gcgaaattgg tggaggtagt ggtggaggta gtgcggccgc cagagatgat ttgaggggac
     2221 gacttgtaac tctcgagaag aaattagcag agcgtaccca agatttgagt cttgcagagt
     2281 ctactttggc aactcgtgag ggtgaattcg aatccctgca aagcaatcta atggagctta
     2341 atgagctccg ggagatgaag gaggatatcg acagaaagaa tgagcaaaca gcagctatat
     2401 tgaagcgaca agcagaccag ataactgaat tagaaactct ttatagggaa gaacaagtcc
     2461 ttcgaaaacg atacttcaac atgatggaag acatgaaagg taagatcagg gtatatgctc
     2521 ggtggagacc cctaagtagt aaggaggtca aagagcggca acaaaacgtg ttgatagcac
     2581 ctgatgaatt caccatcgaa catccttgga aagacgacaa acccaaacaa catcaatttg
     2641 atcacgtttt cgaccaccat gcgacccaag aagaggtctt tgaggataca aagtatttgg
     2701 tccaatcagc aattgacgga tacaatgtct gtatatttgc atacgggcaa actgggtcag
     2761 gcaaaacgtt tacgatttac ggttctgaca acaacccagg tctaactcca cgagctacca
     2821 aagaactttt tggctatctt aagcgagacg ccaataagtt ttcgtttgct ctgaaggttt
     2881 acatgcttga aatatatcaa gattcgctca tcgatctgct tctaccaaag agtgctgcaa
     2941 agccacgaaa gcttgagatt aagaaggact ccaagggaat ggttgtcgta gagaatgcaa
     3001 ctctactgcc gattgcatcc catgatgaac tgcaagccat tgttcataag ggtctggaga
     3061 gacgacatgt ttcgggcacc cacatgaatg cggagagttc acggtcacac ctcattctat
     3121 cagtaatcgt tgaaagcacc aaccgacagt ctcaagttct tgtgaagggc aagctgagtt
     3181 ttgtggatct tgcgggctct gaaagagtga agaaatctgg atccagtggt gaacaactga
     3241 aagaggccca aagcattaat aaatctttgt cagcactagg tgatgtcatt agcgcccttg
     3301 caacagagga gcagcatatt ccttaccgta accacaagct aacaatgctt atgagcgatt
     3361 ctctcggtgg aaatgcgaag accctcatgt ttgtaaatat ctcaccagca gagtctaatc
     3421 tggacgagac ccataattca ctctgctatg ccacaagggt aagatccatc atcaatgagg
     3481 ccagtaaaaa tactacaacg aaagagatac tgcgactgaa gaaacaaatc gcgtactgga
     3541 aggagcaagc gggcaggatg ggagggtccg aggatctgga agaggttatg gatgatcgtg
     3601 gaatcaaaac tgcagattaa acgcgtaccg gtcctagata agtaatgatc ataatcagcc
     3661 atatcacatc tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3721 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3841 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaat tcgtaatcat
     3901 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6061 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6121 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6181 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6241 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     6301 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     6361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     6481 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagttgggat ctttgcattg
     6541 gcccacggct ctcaggatgg ggatgctccc cttcagcacc cggttcccct tggaaactga
     6601 tggtcctggc tctgtggcat ggcagtggca ctgtgaggag ccccctacca gcagcacaca
     6661 gtggggttgg cactgccacg ctccggatgc cgcgctctga tccaacccca taatcaaggg
     6721 aacccgaatt gccccatcat tgcccccacc acccccatcc tgccgggccc tcacacccca
     6781 cgctgccttc tggtgacatt ccccagccca aacccacggc ttcatggcta ccgcggggca
     6841 tttcccattg ccgccccatt atcagctctg cacacctccc gctgtaccca tgcctcgtgg
     6901 ctgcccttct ttgacgtata atcttctaat taatacccgg ccttgtcaaa gtggagcaca
     6961 aacgttaatt aattccccag caggcaggta attaacagtg tgactccctt tttgctgcga
     7021 gtggggctga tacagagaga tgtggcacta tggagcccac ggggtcctgg cactgggtgc
     7081 ccacggaggt ccccatgtgc tgcagtgtca ccgcctccga ggagacagta ttgtccctgc
     7141 ggtgtccctg cagctcagct ctgtccacag ggccacctcc agtttggagg ggacacaatg
     7201 cagccccgat gcaacccatc ctcgcagcat cccagggaca aagaccccac tgcaagaccg
     7261 cacacagggc tgggtcccgc tcccctaata tctacagtgc ttcttgcatg gccccttaat
     7321 caatgcagtt aatcagcatg cgctcatgca ccgctctgga gctgcaaagc ccctcgcagc
     7381 gctgctcacc aacaccgcgc accgccccgg cccagcctgc agcacgcgct gcaaacagga
     7441 aagaaacaaa atattgccca aatgtaggca aaggcattcg gctgccttga cctccgccgg
     7501 gccgggccct gcctgactca gctccttact cagcgctcgc ttcctccctc cggctgccac
     7561 cgccgcagcg cacaccctga caaagagtgg cccttaacgg gctctgaggt gcacccagca
     7621 gtgcactcag cagtccaagg gccggcctgg aggtttgcac cgctacgtgc tgacattagc
     7681 attgaacttg gccctgggta gtgctgcagg ccgggcgggg tgggtgtaga gagtgcagcg
     7741 cgcgttgcac ccggtgcccc ttcccctccc ttgcatccca gcaggctgca ccccagcacc
     7801 aggcccgtgc atgcatgctc ctggtgttat tgcagcctgg tgcatgcatg cgtcttgatg
     7861 gtgcagcgct gtgcatgcat cctccttggt gtgtagcagt ctagtgcatg catacccctc
     7921 ggtgttattg ctgctctgtg cacgcacgct cattgtatca cttcatccca gtgcatgcac
     7981 tcacactgga gcgattgctg ctcggtgcac gcacactcat tgtatcactg cagctcagtg
     8041 gctgcacgca caccggtgtt attgctgctc ggtgcgtgca tgcacatcag tgtcgctgca
     8101 gctcagtgca tgcacgctca ttgcccatcg ctatccctgc ctctcctgct ggcgctcccc
     8161 gggaggtgac ttcaagggga ccgcaggacc acctcggggg tggggggagg gctgcacacg
     8221 cggaccccgc tccccctccc caacaaagca ctgtggaatc aaaaaggggg gaggggggat
     8281 ggaggggcgc gtcacacccc cgccccacac cctcacctcg aggtgagccc cacgttctgc
     8341 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     8401 ttattttgtg cagcgatggg ggcggggggg ggggggcgcg cgccaggcgg ggcggggcgg
     8461 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
     8521 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
     8581 gcgcggcggg cggg
//
