LOCUS       sequence-225112-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SNAP-tagging at NuMA's C-terminal locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  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 6817)
  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: pTK583
FEATURES             Location/Qualifiers
     source          1..6817
                     /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             2783..3328
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     polyA_signal    3449..3570
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3486..3505)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3540..3559
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3601..3634
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3695..4194
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3730..3753)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4127..4146
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4255..5274
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             4255..5274
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    5837..5870
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6423..6440)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6423..6439)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6432..6454)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6648..6667)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6767..6789
                     /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 ggcgccgcta gcatggacaa agactgcgaa atgaagcgca ccaccctgga
     2821 tagccctctg ggcaagctgg aactgtctgg gtgcgaacag ggcctgcacc gtatcatctt
     2881 cctgggcaaa ggaacatctg ccgccgacgc cgtggaagtg cctgccccag ccgccgtgct
     2941 gggcggacca gagccactga tgcaggccac cgcctggctc aacgcctact ttcaccagcc
     3001 tgaggccatc gaggagttcc ctgtgccagc cctgcaccac ccagtgttcc agcaggagag
     3061 ctttacccgc caggtgctgt ggaaactgct gaaagtggtg aagttcggag aggtcatcag
     3121 ctacagccac ctggccgccc tggccggcaa tcccgccgcc accgccgccg tgaaaaccgc
     3181 cctgagcgga aatcccgtgc ccattctgat cccctgccac cgggtggtgc agggcgacct
     3241 ggacgtgggg ggctacgagg gcgggctcgc cgtgaaagag tggctgctgg cccacgaggg
     3301 ccacagactg ggcaagcctg ggctgggtta atctagataa ctgatcataa tcagccatac
     3361 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3421 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     3481 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3541 tggtttgtcc aaactcatca atgtatctta acgcgtcgat catattcaat aacccttaat
     3601 ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag tttacgtcca
     3661 gccaagctta ggatctcgac ctcgaaattc taccgggtag gggaggcgct tttcccaagg
     3721 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     3781 tggcctcgca cacattccac atccaccggt aggcgccaac cgactccgtt ctttggtggc
     3841 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3901 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3961 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     4021 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     4081 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     4141 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc
     4201 atccatctag atctcgagca gctgaagctt accgctagca tggatagatc cggaaagcct
     4261 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     4321 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     4381 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     4441 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     4501 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     4561 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     4621 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     4681 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4741 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4801 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4861 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4921 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4981 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     5041 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     5101 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     5161 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     5221 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataggctagc
     5281 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg
     5341 atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa
     5401 aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt
     5461 gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc
     5521 tttactgaag gctctttact attgctttat gataatgttt catagttgga tatcataatt
     5581 taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct
     5641 atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact
     5701 gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca
     5761 catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag
     5821 atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc
     5881 gaagaggttc actaggatcc ctgtgtcccc gatgctgacc tcacctggtc ctccgcctac
     5941 tgtccctctc agtgccttct ctcagctccc aggccaacag tagccaaacc cctagagaca
     6001 gtgatgcctg cccgcaccct ggcctggtcc ctggtccttc actggcgcct tctcggagct
     6061 ggcccagggg gcctggagca tggacagtgt gggcgctctc cctaccttgc ctcctttttt
     6121 cttaaagcaa agtcacttct ccatcacaac cagatttgag gctggttttg atggctgggt
     6181 ccttgggcct ggccagtctt cctcttagcc tctggatcta gaagggacca taagaggagt
     6241 aggccctggt tcctgctgtc ctggtggctg ggcccagcag gggccctcac tcttgaagtc
     6301 caggactggg tctgacctgg tgggagcacc tgccagagga tgctctttcc caggacggat
     6361 gggccctatg tctcaggagt ggggttgggg gacagccgtt ccatcccaat acgcgtcaat
     6421 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6481 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     6541 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     6601 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     6661 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6721 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6781 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
