LOCUS       sequence-451983-        4009 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-2A-Blast cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4009)
  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 4009)
  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-Blast
FEATURES             Location/Qualifiers
     source          1..4009
                     /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)."
     CDS             1245..1301
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1308..1703
                     /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(1710..1743)
                     /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(1788..1804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1788..1804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1801..1823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1812..1828
                     /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(1836..1842,1843..1860,1861..1866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1881..1902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2019..2036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2190..2778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2270..2289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2949..3740,3741..3809))
                     /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     3572..3591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3810..3914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3982..4000
                     /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 cggagctact aacttcagcc
     1261 tgctgaagca ggccggagac gtggaggaga accctggacc ttctagaatg gccaagcctt
     1321 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     1381 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     1441 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     1501 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     1561 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     1621 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     1681 cctctggtta tgtgtgggag ggccccggga taacttcgta tagcatacat tatacgaacg
     1741 gtagcctcga gaccggtaag agcaagtgaa agcttggcgt aatcatggtc atagctgttt
     1801 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1861 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1921 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1981 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2041 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2101 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2161 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2221 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2281 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2341 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2401 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2461 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2521 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2581 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2641 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2701 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2761 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2821 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2881 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2941 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3001 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3061 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3121 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3181 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3241 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3301 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3361 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3421 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3481 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3541 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3601 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3661 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3721 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3781 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3841 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3901 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     3961 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
