LOCUS       sequence-452010-        3893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty selectable Knock-Out donor cassette. Integrate homology arms 
            to target the N-terminus insertion of a PGK-Blast cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3893)
  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 3893)
  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-KO-PGK-Blast
FEATURES             Location/Qualifiers
     source          1..3893
                     /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"
     promoter        429..928
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(464..487)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     861..880
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    complement(935..968)
                     /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             980..1036
                     /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             1043..1438
                     /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(1445..1478)
                     /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)."
     polyA_signal    1508..1629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1545..1564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1599..1618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1672..1688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1672..1688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1685..1707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1696..1712
                     /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(1720..1726,1727..1744,1745..1750))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1765..1786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1903..1920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2074..2662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2154..2173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2833..3624,3625..3693))
                     /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     3456..3475
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3694..3798)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3866..3884
                     /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 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
      481 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
      541 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
      601 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
      661 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
      721 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
      781 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
      841 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
      901 ctcctcttcc tcatctccgg gcctttcgac gcgttaccgt tcgtatagca tacattatac
      961 gaagttatct ggatccggag ctactaactt cagcctgctg aagcaggccg gagacgtgga
     1021 ggagaaccct ggaccttcta gaatggccaa gcctttgtct caagaagaat ccaccctcat
     1081 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     1141 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     1201 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     1261 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     1321 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     1381 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggccc
     1441 cgggataact tcgtatagca tacattatac gaacggtagc atgggtggcg gttcctgaca
     1501 gacatgaaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1561 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1621 tgtatcttac tcgagaccgg taagagcaag tgaaagcttg gcgtaatcat ggtcatagct
     1681 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1741 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1801 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1861 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1921 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1981 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2041 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2101 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2161 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2221 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2281 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2341 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2401 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2461 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2521 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2581 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2641 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2701 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2761 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2821 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2881 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2941 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3001 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3061 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3121 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3181 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3241 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3301 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3361 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3421 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3481 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3541 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3601 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3661 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3721 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3781 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     3841 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
