LOCUS       sequence-451300-        5750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold-EFS-Puro insertion into the C-term of 
            CEP192 locus. For pericentriolar material visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-CEP192 (Addgene 
            #227288).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5750)
  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 5750)
  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-CEP192
FEATURES             Location/Qualifiers
     source          1..5750
                     /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             994..1671
                     /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             1687..1728
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1750..1783)
                     /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    1905..2026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1942..1961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1996..2015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2076..2287
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2298..2894
                     /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(2298..2317)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2794..2814
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2901..2934)
                     /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(3529..3545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3529..3545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3542..3564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3553..3569
                     /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(3577..3583,3584..3601,3602..3607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3622..3643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3760..3777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3931..4519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4011..4030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4690..5481,5482..5550))
                     /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     5313..5332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5551..5655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5723..5741
                     /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 cgggttggga caagttgttt
      421 taatctttcc agtgcaatat gctgtctgga ctgagtaaaa cgggcattta gggtgttccc
      481 gaaccctttc ttaggtagat agtttttgca ggcagtatac aaagctgtat gtatgatagt
      541 ttagacctct tattttcctt tttatatatt aatagttact ggatagccag gaatacatat
      601 gtatgcaata ctgttttaat ccaattttac gtttttctct cctttgtgtt attttctcat
      661 taaataatag catccaagaa atcataaata catttaagct gaatgtggct tgaccgagct
      721 ctttcctcaa cacctgagcc aggcactgtg ctgcctggga cagggtcacg ggtgctgtca
      781 tgacatgctg ctgtcatgtg cctctctctt tccagagccc agcattacat caacatgccc
      841 gtgcagttca aaccgaagtc cgcaggcaaa tttgaagctt tgcttgtcat tcaaacagat
      901 gaaggcaaga gtattgctat tcgactaatt ggtgaagctc ttagtaaaaa tggaggaagc
      961 ggaggatctg gcggtaccgg aggtggcgga agcatggtgt ctacaggcga ggaactgttt
     1021 accggcgtgg tgcccttcaa gttccagctg aagggcacca tcaacggcaa gagcttcacc
     1081 gtggaaggcg agggcgaggg caatagccac gagggcagcc acaaaggcaa gtatgtgtgc
     1141 accagcggca aactgccaat gtcttgggcc gccctgggaa ctagcttcgg ctatggcatg
     1201 aaatactata ccaagtaccc cagcggcctg aaaaactggt tccacgaggt gatgcctgag
     1261 ggcttcacct acgacagaca catccagtac aagggcgacg gcagcatcca cgccaagcac
     1321 cagcacttca tgaagaacgg cacctaccac aacatcgtgg agttcaccgg ccaggacttc
     1381 aaggagaaca gccccgtgct gaccggcgac atggacgtga gcctgcccaa cgaggtgcag
     1441 cacatcccca ttgatgacgg cgtggagtgc acagtgaccc tgcagtaccc tctgctgagc
     1501 gacgaaagca agtgcgtgga agcctatcag aacaccatca tcaagcccct gcacaatcag
     1561 ccagcccccg atgtgccatt tcactggatc agaaagcagt acacccagag caaggacgac
     1621 accgaggaga gagaccacat catccagagc gagaccctgg aggcccacct gggaggtggc
     1681 ggttccggca agcccatccc caaccccctg ctgggcctgg acagcaccgg aggtggcgga
     1741 tcaacgcgtt accgttcgta tagcatacat tatacgaagt tatctggatc ctgataatga
     1801 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1861 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1921 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1981 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttactag attaggtctt
     2041 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2101 ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg
     2161 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2221 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2281 aacacagggc ctctagaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     2341 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     2401 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     2461 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     2521 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     2581 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     2641 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     2701 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     2761 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     2821 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     2881 gcaagcccgg tgcccccggg ataacttcgt atagcataca ttatacgaac ggtagcctcg
     2941 agaccggtaa gagcaagtga taactagaat acatttttgt gtaaagtaaa ttacataagt
     3001 tgtattttgt taactttatc tttctacact acaattatgc ttttgtatat atattttgta
     3061 tgatggatat ctataattgt agattttgtt tttacaagct aatactgaag actcgactga
     3121 aatattatgt atctagccca tagtattgta cttaactttt acaggtgaga agagagttct
     3181 gtgtttgcat tgattatgat attctgaata aatatggaat atattttaat gtggtatatc
     3241 cagaaatttt tcagtgagct tgtctttttc tatgctgtca tacagaaatt agaatatcac
     3301 tttattctgt taagtatttt ccagcttctg ggtctgttct gtgattttta tcctccatca
     3361 cacagtttcc tttctcctca gaaattcctt ctctgttcct ctgtcgttta gccagttgcc
     3421 cagagtagaa aggacaactt acttttcaag cagctggtta ttttactgag gggaagtctt
     3481 catagtctgt gcaaacacag tgaaggaggt aagcttggcg taatcatggt catagctgtt
     3541 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3601 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3661 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3721 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3781 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3841 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3901 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3961 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4021 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4081 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4141 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4201 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4261 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4321 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4381 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4441 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4501 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4561 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4621 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4681 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4741 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4801 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4861 agcaataaac cagccagccg gaagtgccga gcgcagaagt ggtcctgcaa ctttatccgc
     4921 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4981 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5041 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5101 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5161 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5221 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5281 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5341 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5401 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5461 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5521 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5581 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5641 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5701 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
