LOCUS       sequence-452079-        5748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold-EFS-Puro insertion into the C-terminus 
            of the PLK4 locus. For centriole visualization. To be co-transfected
            with sgRNA plasmid px458-PLK4 (Addgene #227310).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5748)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 5748)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: qTAG-C-mStayGold-EFS-Puro-PLK4
FEATURES             Location/Qualifiers
     source          1..5748
                     /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             993..1670
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     CDS             1686..1727
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1749..1782)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1904..2025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1941..1960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1995..2014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2075..2286
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2297..2893
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2297..2316)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2793..2813
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2900..2933)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3527..3543)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3527..3543)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3540..3562)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3551..3567
                     /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(3575..3581,3582..3599,3600..3605))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3620..3641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3758..3775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3929..4517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4009..4028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4688..5479,5480..5548))
                     /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     5311..5330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5549..5653)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5721..5739
                     /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 ccaccacacc tggctaattt
      421 tgtattttta gtagagacag ggtttcaaca tattggccag gctggtctca aacacctgac
      481 ctcgtaatcc accccccttg gcctcccaaa gtgctgggat tacaggcatt aaccaccgcg
      541 cctgtccggg gccttttaaa tttaatgagg aatccagatt tttggcttgc ttttgaaatt
      601 acgaagccag ttcaagttta tctaattttc cttctgttac ttaaatgtgt ttagaaaatg
      661 gtctgtttta aaagaaaaga aactgtatgc ttcacttgat ttaggtatct aataaataat
      721 tgttataatt tttctgaggt gcctcttgtt attgaagaat tactgaaaaa tataatgtgc
      781 tttttgggac tctcatgaga accaagtaat tcagttacga ttttgtcttt ttttcttttg
      841 cttcacttag gtatggagaa aatgaaaaat taccagacta catcaaacag aaattacagt
      901 gtctgtcttc catccttttg atgttttcta atccgactcc taattttcat ggaggaagcg
      961 gaggatctgg cggtaccgga ggtggcggaa gcatggtgtc tacaggcgag gaactgttta
     1021 ccggcgtggt gcccttcaag ttccagctga agggcaccat caacggcaag agcttcaccg
     1081 tggaaggcga gggcgagggc aatagccacg agggcagcca caaaggcaag tatgtgtgca
     1141 ccagcggcaa actgccaatg tcttgggccg ccctgggaac tagcttcggc tatggcatga
     1201 aatactatac caagtacccc agcggcctga aaaactggtt ccacgaggtg atgcctgagg
     1261 gcttcaccta cgacagacac atccagtaca agggcgacgg cagcatccac gccaagcacc
     1321 agcacttcat gaagaacggc acctaccaca acatcgtgga gttcaccggc caggacttca
     1381 aggagaacag ccccgtgctg accggcgaca tggacgtgag cctgcccaac gaggtgcagc
     1441 acatccccat tgatgacggc gtggagtgca cagtgaccct gcagtaccct ctgctgagcg
     1501 acgaaagcaa gtgcgtggaa gcctatcaga acaccatcat caagcccctg cacaatcagc
     1561 cagcccccga tgtgccattt cactggatca gaaagcagta cacccagagc aaggacgaca
     1621 ccgaggagag agaccacatc atccagagcg agaccctgga ggcccacctg ggaggtggcg
     1681 gttccggcaa gcccatcccc aaccccctgc tgggcctgga cagcaccgga ggtggcggat
     1741 caacgcgtta ccgttcgtat agcatacatt atacgaagtt atctggatcc tgataatgat
     1801 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     1861 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     1921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttactaga ttaggtcttg
     2041 aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2101 cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg
     2161 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2221 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2281 acacagggcc tctagaatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     2341 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     2401 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     2461 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     2521 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     2581 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     2641 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     2701 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     2761 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     2821 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     2881 caagcccggt gcccccggga taacttcgta tagcatacat tatacgaacg gtagcctcga
     2941 gaccggtaag agcaagtgat gattaaaact cgtttcagac atataagttt aataaataac
     3001 ttttttgttg actttcaagt aaagtgattt tttttaattt aacataaagt cttcagaaag
     3061 cctttctatg aaagaatttt aacctataat gtaaaggatg tattctgaga gaacaaagca
     3121 gaatgaaact tgagtcactt actaaatata gtggatataa aatagaacac ctgactttgc
     3181 tcttagacca taacccccga acttactatg ttcatatatt tgtattgaac aatcttttaa
     3241 aagcaaaaat gtaaatgatg tgtagtttat ttgtgctttt attgttttcc ctgcgtctca
     3301 gacatgttga gaatcatgga caaaacctgc tggaattttg gaatttttga agatgtaaat
     3361 aatgtgtatt tatgttataa gtaacatatg taaacatgta tatttgtttt atatttattt
     3421 ttgtaacacc agtgtctgat gaaacatttt tgcaaatgca ttttataaaa aaataaatat
     3481 agtgataagt tacattatct tttgattcaa gcttggcgta atcatggtca tagctgtttc
     3541 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3601 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3661 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3721 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3781 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3841 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3901 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3961 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4021 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4081 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4141 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4201 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4261 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4321 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4381 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4441 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4501 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4561 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4621 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4681 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4741 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4801 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4861 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4921 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4981 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5041 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5101 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5161 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5221 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5281 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5341 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5401 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5461 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5521 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5581 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5641 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     5701 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
