LOCUS       sequence-223939-        7194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP-Nano tagging at NuMA's C-terminal locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7194)
  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 7194)
  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: pTK421
FEATURES             Location/Qualifiers
     source          1..7194
                     /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"
     regulatory      2825..2834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2831..3562
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             3638..3967
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    4095..4216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4132..4151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4186..4205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4223..4678)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4360..4381)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4572..4591
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4705..4809
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4811..5168
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4811..5002
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4834..4854)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5019..5154
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5203..5997
                     /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             5203..5997
                     /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(5257..5276)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5867..5886
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6185..6204)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(6800..6817)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6800..6816)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6809..6831)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7025..7044)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7144..7166
                     /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 atccaccgga ggtggcggat caggaggtgg aagtggaggt ggctctgccg gtggaggtgg
     2821 atctgccacc atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct
     2881 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
     2941 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     3001 cgccttcgac atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac
     3061 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
     3121 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     3181 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat
     3241 gcagaagaaa acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg
     3301 cctggaaggc agaaccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa
     3361 cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta
     3421 ctatgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
     3481 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa
     3541 tggcatggac gagctgtaca agtccggact cagatctcgc gctcaagctt cgaacgaatt
     3601 cggcattgat ctgagcggcc tgaccctgca ggaattcagc tccccgaaac gccctaagct
     3661 gctgcgtgaa tattacgatt ggctggttga taacagcttt accccatatc tggtggtgga
     3721 tgccacatac ctgggcgtga acgtgcccgt ggagtatgtg aaagacggtc agatcgtgct
     3781 gaatctgtct gcaagtgcga ccggcaacct gcaactgaca aatgatttta tccagttcaa
     3841 cgcccgcttt aagggcgtgt ctcgtgaact gtatatcccg atgggtgccg ctctggccat
     3901 ttacgctcgc gagaacggcg atggtgtgat gttcgaacca gaagaaatct atgacgagct
     3961 gaatattggt taagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     4021 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     4081 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4141 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4201 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     4261 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     4321 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     4381 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     4441 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     4501 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4561 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4621 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     4681 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4741 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4801 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     4861 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4921 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4981 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     5041 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     5101 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     5161 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     5221 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5281 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5341 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     5401 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5461 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5521 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5581 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5641 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     5701 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     5761 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     5821 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     5881 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     5941 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     6001 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     6061 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     6121 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     6181 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     6241 gacagaataa aacgcacggt gttgggtcgt tggatccctg tgtccccgat gctgacctca
     6301 cctggtcctc cgcctactgt ccctctcagt gccttctctc agctcccagg ccaacagtag
     6361 ccaaacccct agagacagtg atgcctgccc gcaccctggc ctggtccctg gtccttcact
     6421 ggcgccttct cggagctggc ccagggggcc tggagcatgg acagtgtggg cgctctccct
     6481 accttgcctc cttttttctt aaagcaaagt cacttctcca tcacaaccag atttgaggct
     6541 ggttttgatg gctgggtcct tgggcctggc cagtcttcct cttagcctct ggatctagaa
     6601 gggaccataa gaggagtagg ccctggttcc tgctgtcctg gtggctgggc ccagcagggg
     6661 ccctcactct tgaagtccag gactgggtct gacctggtgg gagcacctgc cagaggatgc
     6721 tctttcccag gacggatggg ccctatgtct caggagtggg gttgggggac agccgttcca
     6781 tcccaatacg cgtcaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6841 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6901 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     6961 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     7021 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     7081 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     7141 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcga
//
