LOCUS       sequence-451990-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty C-terminus donor cassette. Integrate homology arms to target 
            the C-terminus insertion of a mStayGold-EFS-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4650)
  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 4650)
  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
FEATURES             Location/Qualifiers
     source          1..4650
                     /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             444..1121
                     /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             1137..1178
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1200..1233)
                     /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    1355..1476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1392..1411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1446..1465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1526..1737
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1748..2344
                     /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(1748..1767)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2244..2264
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2351..2384)
                     /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(2429..2445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2429..2445)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2442..2464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2453..2469
                     /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(2477..2483,2484..2501,2502..2507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2522..2543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2660..2677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2831..3419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2911..2930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3590..4381,4382..4450))
                     /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     4213..4232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4451..4555)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4623..4641
                     /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 cggaggaagc ggaggatctg
      421 gcggtaccgg aggtggcgga agcatggtgt ctacaggcga ggaactgttt accggcgtgg
      481 tgcccttcaa gttccagctg aagggcacca tcaacggcaa gagcttcacc gtggaaggcg
      541 agggcgaggg caatagccac gagggcagcc acaaaggcaa gtatgtgtgc accagcggca
      601 aactgccaat gtcttgggcc gccctgggaa ctagcttcgg ctatggcatg aaatactata
      661 ccaagtaccc cagcggcctg aaaaactggt tccacgaggt gatgcctgag ggcttcacct
      721 acgacagaca catccagtac aagggcgacg gcagcatcca cgccaagcac cagcacttca
      781 tgaagaacgg cacctaccac aacatcgtgg agttcaccgg ccaggacttc aaggagaaca
      841 gccccgtgct gaccggcgac atggacgtga gcctgcccaa cgaggtgcag cacatcccca
      901 ttgatgacgg cgtggagtgc acagtgaccc tgcagtaccc tctgctgagc gacgaaagca
      961 agtgcgtgga agcctatcag aacaccatca tcaagcccct gcacaatcag ccagcccccg
     1021 atgtgccatt tcactggatc agaaagcagt acacccagag caaggacgac accgaggaga
     1081 gagaccacat catccagagc gagaccctgg aggcccacct gggaggtggc ggttccggca
     1141 agcccatccc caaccccctg ctgggcctgg acagcaccgg aggtggcgga tcaacgcgtt
     1201 accgttcgta tagcatacat tatacgaagt tatctggatc ctgataatga tcataatcag
     1261 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1321 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     1381 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1441 tagttgtggt ttgtccaaac tcatcaatgt atcttactag attaggtctt gaaaggagtg
     1501 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     1561 ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg
     1621 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     1681 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagggc
     1741 ctctagaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     1801 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     1861 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     1921 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     1981 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     2041 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     2101 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     2161 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     2221 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     2281 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     2341 tgcccccggg ataacttcgt atagcataca ttatacgaac ggtagcctcg agaccggtaa
     2401 gagcaagtga aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2461 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2521 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2581 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2641 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2701 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2761 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2821 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2881 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2941 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3001 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3061 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3241 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3721 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4441 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4501 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4561 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     4621 aaaataggcg tatcacgagg ccctttcgtc
//
