LOCUS       sequence-225211-        7005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mAID-mClover-3FLAG tagging at NuMA's C-terminal locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7005)
  AUTHORS   Okumura M, Natsume T, Kanemaki MT, Kiyomitsu T
  TITLE     Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling 
            forces as a multi-arm ensemble.
  JOURNAL   Elife. 2018 May 31;7. pii: 36559. doi: 10.7554/eLife.36559.
  PUBMED    29848445
REFERENCE   2  (bases 1 to 7005)
  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: "Elife."; 
            date: "2018-05-31"; volume: "7. pii: 36559. doi"; pages: " 
            10.7554/eLife.36559"
COMMENT     Sequence Label: pTK398
FEATURES             Location/Qualifiers
     source          1..7005
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2777..2980
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(2987..2989,2990..2992,2993..3703)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3032..3053)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3640..3661
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3716..3781
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3906..4027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3943..3962)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3997..4016
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4034..4489)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4171..4192)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4383..4402
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4516..4620
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4622..4979
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4622..4813
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4645..4665)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4830..4965
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5014..5808
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5014..5808
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5068..5087)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5678..5697
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5996..6015)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(6611..6628)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6611..6627)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6620..6642)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6836..6855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6955..6977
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc ggcgcgccat tgggatggaa cgagcctcaa agaaggccct
     2281 gtccaaggct tcccccaaca ctcgcagtgg aacccgccgt tctccgcgca ttgccaccac
     2341 cacagccagc gccgccactg ctgccgccat tggtgccacc cctcgagcca agggcaaggt
     2401 agaggcactg gcaggatggg ggagggaaac ccgatgagag catgggcacc ccccagaccg
     2461 ccccaccaag tagcatccaa gatggggcca cgccaggctg ctggagggtg tggggtaaga
     2521 ggtgtccctg tcctggtagc tcctctccaa ggagagaaga cagatggccc cattctcatg
     2581 gagagcaccc agagctgctc aggcacagcc agcaactgat aaccctgggg ctcacaaggg
     2641 aggggaacca gacaggggcg tgggagcagg aagagatctt cccagccagt gggtgcaggc
     2701 tagtatcgca agttctcatc tggccatgtg actgtgtctc tttcaggcaa agcacggagg
     2761 atccggtgca ggcgccaagg agaagagtgc ttgtcctaaa gatccagcca aacctccggc
     2821 caaggcacaa gttgtgggat ggccaccggt gagatcatac cggaagaacg tgatggtttc
     2881 ctgccaaaaa tcaagcggtg gcccggaggc ggcggcgttc gtgaaggtat caatggacgg
     2941 agcaccgtac ttgaggaaaa tcgatttgag gatgtataaa gctagcatgg tgagcaaggg
     3001 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3061 ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     3121 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
     3181 cggctacggc gtggcctgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3241 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atctctttca aggacgacgg
     3301 tacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3361 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3421 cttcaacagc cacaacgtct atatcacggc cgacaagcag aagaacggca tcaaggctaa
     3481 cttcaagatc cgccacaacg ttgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3541 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagccatca
     3601 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3661 gaccgccgcc gggattacac atggcatgga cgagctgtac aagggaggag gatcggacta
     3721 caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa
     3781 gtaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     3841 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3901 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3961 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4021 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     4081 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     4141 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4201 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4261 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4321 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4381 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4441 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     4501 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4561 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4621 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4681 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4741 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4801 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4861 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4921 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     4981 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     5041 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     5101 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     5161 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     5221 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     5281 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     5341 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5401 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5461 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5521 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     5581 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5641 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5701 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5761 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5821 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5881 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5941 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     6001 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     6061 aaacgcacgg tgttgggtcg ttggatccct gtgtccccga tgctgacctc acctggtcct
     6121 ccgcctactg tccctctcag tgccttctct cagctcccag gccaacagta gccaaacccc
     6181 tagagacagt gatgcctgcc cgcaccctgg cctggtccct ggtccttcac tggcgccttc
     6241 tcggagctgg cccagggggc ctggagcatg gacagtgtgg gcgctctccc taccttgcct
     6301 ccttttttct taaagcaaag tcacttctcc atcacaacca gatttgaggc tggttttgat
     6361 ggctgggtcc ttgggcctgg ccagtcttcc tcttagcctc tggatctaga agggaccata
     6421 agaggagtag gccctggttc ctgctgtcct ggtggctggg cccagcaggg gccctcactc
     6481 ttgaagtcca ggactgggtc tgacctggtg ggagcacctg ccagaggatg ctctttccca
     6541 ggacggatgg gccctatgtc tcaggagtgg ggttggggga cagccgttcc atcccaatac
     6601 gcgtcaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     6661 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     6721 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     6781 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     6841 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     6901 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6961 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcga
//
