LOCUS       sequence-223933-        5373 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Nano-mCherry tagging at DHC's N-terminal locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5373)
  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 5373)
  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: pTK426
FEATURES             Location/Qualifiers
     source          1..5373
                     /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"
     regulatory      875..884
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             881..1276
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1289..1345
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1349..1678
                     /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"
     regulatory      1706..1715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1712..2419
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1857..1875)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2125..2144
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2465..2485
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2498..2542
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3152..3168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3152..3168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3165..3187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3176..3192
                     /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(3200..3206,3207..3224,3225..3230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3383..3400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3554..4142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3634..3653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4313..5104,5105..5173))
                     /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     4936..4955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5174..5278)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5346..5364
                     /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 tgctattcgc tgcgtccaca gcagtgaaga agacagacgc agcccctgac caacctcgcc
      481 aagcccgggc gccgagccag gacgttgagc caagtcccca tctcctagga ctcgcagtgc
      541 ggaggctgcg ggccccgacc agcgccgaga ctacaagtcc cagaagggcg cgcggcgaca
      601 tcgccgggcc cgccccccgc gcacgcgcgg cgccgtgacg ctaaggccgc gagcgagcgc
      661 gggtggctgc ggcggctgcg gccggcgggg cggggcgggg ccacctggca gtggctgcct
      721 ttagggcgga gccggctccc gctctcctca gtctgcggtg ggctagcgga cggtccggct
      781 tccggcggcc gtttctgtct cttgctggct gtctcgctga gtcgcggccg ccttctcatc
      841 gctcctggaa ggtcccgagc gcgacaccgg atccgccacc atggccaagc ctttgtctca
      901 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
      961 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     1021 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     1081 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     1141 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     1201 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     1261 ttatgtgtgg gagggctcag gaagcggagc tactaacttc agcctgctga agcaggctgg
     1321 agacgtggag gagaaccctg gacctatgag ctccccgaaa cgccctaagc tgctgcgtga
     1381 atattacgat tggctggttg ataacagctt taccccatat ctggtggtgg atgccacata
     1441 cctgggcgtg aacgtgcccg tggagtatgt gaaagacggt cagatcgtgc tgaatctgtc
     1501 tgcaagtgcg accggcaacc tgcaactgac aaatgatttt atccagttca acgcccgctt
     1561 taagggcgtg tctcgtgaac tgtatatccc gatgggtgcc gctctggcca tttacgctcg
     1621 cgagaacggc gatggtgtga tgttcgaacc agaagaaatc tatgacgagc tgaatattgg
     1681 tggaggtgga tctggcggcg gtagcgccac catggtgagc aagggcgagg aggataacat
     1741 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1801 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1861 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1921 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1981 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     2041 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     2101 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     2161 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     2221 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     2281 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     2341 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2401 catggacgag ctgtacaagt ccggagggga tcggtggagc tccaccggtg gcggccgctc
     2461 tagagagaat ctttattttc agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt
     2521 cgaacgccag cacatggaca gcggaggtgg aggtactagt ggatccatgt cggagcccgg
     2581 gggcggcggc ggcgaagatg gtagtgcagg tctagaagtg tcggccgtgc agaatgtggc
     2641 ggacgtgtcg gtgctgcaga agcacctgcg caagctggtg ccgctgctgc tggaggacgg
     2701 cggcgaggcg ccggccgcgc tggaggcggc gctggaggag aagagcgccc tggagcagat
     2761 gcgcaagttc ctttcggacc cgcaggtcca cacggtgctg gtggagcgct ccacgctcaa
     2821 aggtgcgggg ccgcggaggg cagggtcgcc agagccaggc ctcgcggaat gcagggcctg
     2881 ccaggtcctc cggggtcgca gatgtccccg ggatgggagg agcccggcag ctgcagatga
     2941 cccctggatg ggcagagccc ggcggccgca gacgtcccgc cggccgggtc cccagggccg
     3001 cagacgcccc gcagaggccg gcgcggcgcc cggggctcgc cctcgccaca cccactgccc
     3061 ggcccggagc ccccagggcg ccccgctcct ggtcgcgggg aggggaaagg gggttccatc
     3121 ccaatggcgc gccgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3181 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3301 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3361 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3421 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3481 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3541 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3601 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3661 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3721 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3781 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3841 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3901 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3961 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4021 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4081 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4141 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4201 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4261 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4321 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4381 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4441 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4501 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4561 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4621 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4681 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4741 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4801 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4861 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4921 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4981 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5041 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5101 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5161 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5281 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5341 ataaaaatag gcgtatcacg aggccctttc gtc
//
