LOCUS       sequence-451996-        4425 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-Zeo cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4425)
  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 4425)
  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-Zeo
FEATURES             Location/Qualifiers
     source          1..4425
                     /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..2119
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     protein_bind    complement(2126..2159)
                     /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(2204..2220)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2204..2220)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2217..2239)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2228..2244
                     /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(2252..2258,2259..2276,2277..2282))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2297..2318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2435..2452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2606..3194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2686..2705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3365..4156,4157..4225))
                     /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     3988..4007
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4226..4330)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4398..4416
                     /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 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     1801 agcggtcgag ttctggaccg accggctcgg gttcagccgg gacttcgtgg aggacgactt
     1861 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     1921 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     1981 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     2041 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     2101 cgtggccgag gagcaggacc ccgggataac ttcgtatagc atacattata cgaacggtag
     2161 cctcgagacc ggtaagagca agtgaaagct tggcgtaatc atggtcatag ctgtttcctg
     2221 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2281 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2341 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2401 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3001 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3061 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3121 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3181 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3241 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3301 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3361 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3421 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3481 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3541 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3601 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3661 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3721 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3781 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3841 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3901 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3961 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4021 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4081 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4141 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4201 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4261 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4321 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4381 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
