LOCUS       sequence-216391-        6252 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SNAP-tagging at p150's C-terminal locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6252)
  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 6252)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pTK587
FEATURES             Location/Qualifiers
     source          1..6252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             674..1219
                     /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    1340..1461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1377..1396)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1431..1450
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1492..1525
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1586..2085
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1621..1644)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2018..2037
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2146..3165
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2146..3165
                     /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    3728..3761
                     /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(4031..4047)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4031..4047)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4044..4066)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4055..4071
                     /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(4079..4085,4086..4103,4104..4109))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4124..4145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4262..4279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4433..5021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4513..4532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5192..5983,5984..6052))
                     /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     5815..5834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6053..6157)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6225..6243
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac
      421 gccttggtct cagtgctcag ggaccccttc agtggcccac agctcaagtg agcccctgac
      481 ctggagtctt tttctcctca tcaaccacca ggccaaggag gagcagcagg atgacacagt
      541 ctacatgggc aaagtgacct tctcatgtgc ggctggtttt ggacagcgac accggctggt
      601 gctgacccag gagcagctgc atcaattgca ttcacgattg ataagtggag gatccggtgc
      661 aggcgccgct agcatggaca aagactgcga aatgaagcgc accaccctgg atagccctct
      721 gggcaagctg gaactgtctg ggtgcgaaca gggcctgcac cgtatcatct tcctgggcaa
      781 aggaacatct gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc
      841 agagccactg atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat
      901 cgaggagttc cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg
      961 ccaggtgctg tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctacagcca
     1021 cctggccgcc ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg
     1081 aaatcccgtg cccattctga tcccctgcca ccgggtggtg cagggcgacc tggacgtggg
     1141 gggctacgag ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact
     1201 gggcaagcct gggctgggtt aatctagata actgatcata atcagccata ccacatttgt
     1261 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     1321 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     1381 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1441 caaactcatc aatgtatctt aacgcgtcga tcatattcaa taacccttaa tataacttcg
     1501 tataatgtat gctatacgaa gttattaggt ctgaagagga gtttacgtcc agccaagctt
     1561 aggatctcga cctcgaaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     1621 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     1681 acacattcca catccaccgg taggcgccaa ccgactccgt tctttggtgg ccccttcgcg
     1741 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     1801 aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct
     1861 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     1921 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     1981 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     2041 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg catccatcta
     2101 gatctcgagc agctgaagct taccgctagc atggatagat ccggaaagcc tgaactcacc
     2161 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2221 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2281 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     2341 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     2401 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     2461 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     2521 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     2581 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     2641 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     2701 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     2761 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     2821 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     2881 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     2941 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3001 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3061 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3121 agtggaaacc gacgccccag cactcgtccg agggcaaagg aataggctag ccgcccgccc
     3181 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcgat gatatcagat
     3241 ccccgggatg cagaaattga tgatctatta aacaataaag atgtccacta aaatggaagt
     3301 ttttcctgtc atactttgtt aagaagggtg agaacagagt acctacattt tgaatggaag
     3361 gattggagct acgggggtgg gggtggggtg ggattagata aatgcctgct ctttactgaa
     3421 ggctctttac tattgcttta tgataatgtt tcatagttgg atatcataat ttaaacaagc
     3481 aaaaccaaat taagggccag ctcattcctc ccactcatga tctatagatc tatagatctc
     3541 tcgtgggatc attgtttttc tcttgattcc cactttgtgg ttctaagtac tgtggtttcc
     3601 aaatgtgtca gtttcatagc ctgaagaacg agatcagcag cctctgttcc acatacactt
     3661 cattctcagt attgttttgc caagttctaa ttccatcaga agctggtcga gatccggaac
     3721 ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtccct cgaagaggtt
     3781 cactaggatc cgcactcctt tcccctgctg tccccttcga ccctcagccc tctggtgccg
     3841 ctctgcccga tgcacagcca cctcagccag cccccaggta gaaacgtggg ttaagctctt
     3901 cctgccccgt tcagcttcac tcccaccctt tcagcgtcct gccccttcac cttgacccgg
     3961 gttcccccac tcccattccc tggcctctgc cgttccatcc caatggcgcg ccgagcttgg
     4021 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4081 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4141 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4201 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4261 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4321 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4381 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4441 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4501 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4561 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4621 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4681 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4741 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4801 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4861 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4921 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4981 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5041 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5101 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5161 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5221 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5281 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5341 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5401 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5461 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5521 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5581 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5641 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5701 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5761 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5821 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5881 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5941 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6001 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6061 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6121 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6181 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     6241 ggccctttcg tc
//
