LOCUS       sequence-413747-        3847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Blast-2A-ultraID cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3847)
  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 3847)
  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-N-Blast-ultraID
FEATURES             Location/Qualifiers
     source          1..3847
                     /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"
     protein_bind    complement(423..456)
                     /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             465..860
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             870..926
                     /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"
     protein_bind    complement(933..966)
                     /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)."
     CDS             990..1031
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1626..1642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1626..1642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1639..1661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1650..1666
                     /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(1674..1680,1681..1698,1699..1704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1719..1740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1857..1874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2028..2616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2108..2127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2787..3578,3579..3647))
                     /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     3410..3429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3648..3752)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3820..3838
                     /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 cggcaagagc aagaccggta
      421 gctaccgttc gtatagcata cattatacga agttatctgc tagcatggcc aagcctttgt
      481 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      541 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      601 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      661 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      721 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
      781 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      841 ctggttatgt gtgggagggc ggatccggag ctactaactt cagcctgctg aagcaggccg
      901 gagacgtgga ggagaaccct ggaccttcta gaataacttc gtatagcata cattatacga
      961 acggtagcac gcgtggaggt ggcggttccg gcaagcccat ccccaacccc ctgctgggcc
     1021 tggacagcac cggaggtggc ggatcaatgt tcaagaacct gatctggctg aaggaggtgg
     1081 acagcaccca ggagagactg aaggagtgga acgtgtccta cggcaccgcc ctggtggccg
     1141 acagacagac caagggcaga ggcggccccg gcagaaagtg gctgagccag gagggcggcc
     1201 tgtacttcag cttcctgctg aaccccaagg agttcgagaa cctgctgcag ctgcccctgg
     1261 tgctgggcct gagcgtgagc gaggccctgg aggagatcac cgagatcccc ttcagcctga
     1321 agtggcccaa cgacgtgtac ttccaggaga agaaggtgag cggcgtgctg tgcgagctga
     1381 gcaaggacaa gctgatcgtg ggcatcggca tcaacgtgaa ccagagagag atccccgagg
     1441 agatcaagga cagagccacc accctgtacg agatcaccgg caaggactgg gacagaaagg
     1501 aggtgctgct gaaggtgctg aagagaatca gcgagaacct gaagaagttc aaggagaagg
     1561 gtggaggcgg atctctcgag ggaggaagcg gaggatctgg cggtaccaag cttggcgtaa
     1621 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1681 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1741 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1801 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1861 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1921 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1981 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2041 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2101 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2161 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2221 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2281 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2341 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2401 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2461 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2521 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2581 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2641 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2701 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2761 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2821 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2881 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2941 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3001 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3061 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3121 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3181 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3241 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3301 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3361 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3421 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3481 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3541 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3601 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3661 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3721 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3781 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3841 tttcgtc
//
