LOCUS       sequence-413743-        4030 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-mScarlet cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4030)
  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 4030)
  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-mScarlet
FEATURES             Location/Qualifiers
     source          1..4030
                     /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"
     CDS             1047..1742
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1180..1198)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1448..1467
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1809..1825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1809..1825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1822..1844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1833..1849
                     /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(1857..1863,1864..1881,1882..1887))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1902..1923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2040..2057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2211..2799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2291..2310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2970..3761,3762..3830))
                     /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     3593..3612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3831..3935)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4003..4021
                     /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 ggatcaatgg tgagcaaggg cgaggcagtg atcaaggagt
     1081 tcatgcggtt caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg
     1141 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     1201 gtggccccct gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg
     1261 ccttcaccaa gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct
     1321 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca
     1381 cctccctgga ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc
     1441 ctgacggccc cgtaatgcag aagaagacaa tgggctggga agcgtccacc gagcggttgt
     1501 accccgagga cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg
     1561 gccgctacct ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg
     1621 gcgcctacaa cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg
     1681 tggaacagta cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     1741 agggtggagg cggatctctc gagggaggaa gcggaggatc tggcggtacc aagcttggcg
     1801 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1861 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1921 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1981 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2041 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2101 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2161 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2221 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2281 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2341 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2401 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2461 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2521 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2581 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2641 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2701 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2761 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2821 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2881 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2941 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3001 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3061 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3121 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3181 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3241 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3301 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3361 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3421 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3481 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3541 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3601 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3661 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3721 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3781 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3841 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3901 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3961 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4021 ccctttcgtc
//
