LOCUS       sequence-413742-        4051 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-moxGFP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4051)
  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 4051)
  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-moxGFP
FEATURES             Location/Qualifiers
     source          1..4051
                     /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             join(1047..1049,1050..1052,1053..1763)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1092..1113)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1700..1721
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1830..1846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1830..1846)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1843..1865)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1854..1870
                     /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(1878..1884,1885..1902,1903..1908))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1923..1944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2061..2078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2232..2820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2312..2331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2991..3782,3783..3851))
                     /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     3614..3633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3852..3956)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4024..4042
                     /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 cgaggagctg ttcaccgggg
     1081 tggtgcccat cctggtcgag ctggacggcg acgtgaacgg ccacaagttc tccgtgcggg
     1141 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc agcaccaccg
     1201 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagagct
     1261 tctcccgcta ccccgaccac atgaagcgcc acgacttctt caagagcgcc atgcccgaag
     1321 gctacgtcca ggagcgcacc atctccttca aggacgacgg cacctacaag acccgcgccg
     1381 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1441 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaactcc cacaacgtct
     1501 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccataacg
     1561 tggaggacgg ctccgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1621 gccccgtgct gctgcccgac aaccactacc tgtccaccca gtccaagctg tccaaagacc
     1681 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1741 acggcatgga cgagctgtac aagggtggag gcggatctct cgagggagga agcggaggat
     1801 ctggcggtac caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1861 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1921 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1981 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2041 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2101 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2161 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2221 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2281 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2341 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2401 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2461 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2521 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2581 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2641 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2701 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2761 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2821 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2881 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2941 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3001 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3061 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3121 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3181 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3241 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3301 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3361 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3421 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3481 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3541 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3601 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3661 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3721 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3781 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3841 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3901 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3961 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4021 aaaaataggc gtatcacgag gccctttcgt c
//
