LOCUS       sequence-451993-        4449 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-Blast cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4449)
  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 4449)
  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-Blast
FEATURES             Location/Qualifiers
     source          1..4449
                     /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..2143
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(2150..2183)
                     /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(2228..2244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2228..2244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2241..2263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2252..2268
                     /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(2276..2282,2283..2300,2301..2306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2321..2342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2459..2476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2630..3218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2710..2729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3389..4180,4181..4249))
                     /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     4012..4031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4250..4354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4422..4440
                     /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 gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     1801 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     1861 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     1921 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     1981 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     2041 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     2101 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggccccggga taacttcgta
     2161 tagcatacat tatacgaacg gtagcctcga gaccggtaag agcaagtgaa agcttggcgt
     2221 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2281 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4381 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4441 cctttcgtc
//
