LOCUS       sequence-413792-        5137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-moxGFP insertion into the N-terminus of 
            the TUBA1B locus for tubulin visualization. To be co-transfected 
            with sgRNA plasmid px330-PITCh-TUBA1B Addgene #207763.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5137)
  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 5137)
  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-TUBA1B
FEATURES             Location/Qualifiers
     source          1..5137
                     /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(981..1014)
                     /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             1023..1418
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1428..1484
                     /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(1491..1524)
                     /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             1533..1559
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1533..1559
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1563..1589
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1599..1601,1602..1604,1605..2315)
                     /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(1644..1665)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2252..2273
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2916..2932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2916..2932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2929..2951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2940..2956
                     /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(2964..2970,2971..2988,2989..2994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3009..3030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3147..3164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3318..3906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3398..3417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4077..4868,4869..4937))
                     /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     4700..4719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4938..5042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5110..5128
                     /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 cctgcccgga aatgtgacca
      421 cctagtctaa agtgggcttc tggggcctga gcgctggatg gatgcccacc ttcctgtctt
      481 ggtcctccaa aggaggaagc tgtgactgag ctgtcttggt ctggaaggag gccttcccgg
      541 tttaggatgg gaaggtaaca ttcattaaaa gcaacgtaga ctatagtgta gctgttctca
      601 aaagtagtac atcttagaaa aggatcttta gaaaagatcg ctttagaaaa ggaaattcgt
      661 tttcagatta cgtgagtagc ctaggtaaca cagccagacc tcatctccac aaaaaaaatg
      721 aaaaaattag ccagcttggt ggtctgtgcc tgtggtccca gctgctccag aggctgaggt
      781 ggggggatga ctggagccta ggctgcagtg agcctagatg gcatcactgc actcaagact
      841 gggcgacaga ccttatctct aaaaaaataa agattgcatg agtattttgt tccacttgac
      901 agtcatcaat agattggttt aaattgtgat atctttttta cttcccgcag agagaatgtg
      961 gcaagagcaa gaccggtagc taccgttcgt atagcataca ttatacgaag ttatctgcta
     1021 gcatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     1081 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     1141 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     1201 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     1261 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     1321 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     1381 ttcgtgaatt gctgccctct ggttatgtgt gggagggcgg atccggagct actaacttca
     1441 gcctgctgaa gcaggccgga gacgtggagg agaaccctgg accttctaga ataacttcgt
     1501 atagcataca ttatacgaac ggtagcacgc gttaccccta cgacgtgccc gactacgccg
     1561 gctatccgta tgatgtcccg gactatgcag gaagcggaat ggtgagcaag ggcgaggagc
     1621 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtgaac ggccacaagt
     1681 tctccgtgcg gggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     1741 tcagcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1801 gcgtgcagag cttctcccgc taccccgacc acatgaagcg ccacgacttc ttcaagagcg
     1861 ccatgcccga aggctacgtc caggagcgca ccatctcctt caaggacgac ggcacctaca
     1921 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1981 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaact
     2041 cccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     2101 tccgccataa cgtggaggac ggctccgtgc agctcgccga ccactaccag cagaacaccc
     2161 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgtccacc cagtccaagc
     2221 tgtccaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2281 ccgggatcac tcacggcatg gacgagctgt acaagctcga gggaggaagc ggaggatctg
     2341 gcggtaccga gtgcatctcc atccacgttg gccaggctgg tgtccagatt ggcaatgcct
     2401 gctgggagct ctactgcctg gaacacggca tccagcccga tggccagatg ccaagtgaca
     2461 agaccattgg gggaggagat gactccttca acaccttctt cagtgagacg ggcgctggca
     2521 agcacgtgcc ccgggctgtg tttgtagact tggaacccac agtcattggt gagttgacct
     2581 cagtaacctg agatcccagg atgctgggac aggaggtctg tccaggggct tctcttgtca
     2641 ctcactcact ccctccgtcc ttctctccct cctccagatg aagttcgcac tggcacctac
     2701 cgccagctct tccaccctga gcagctcatc acaggcaagg aagatgctgc caataactat
     2761 gcccgagggc actacaccat tggcaaggag atcattgacc ttgtgttgga ccgaattcgc
     2821 aagctggtaa gcaccacata taaatatgca tttaatgtgg tgtgatagtt ccagtgcaag
     2881 ttgggtggag tgactgaaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2941 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3001 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3061 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3121 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3181 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3241 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3301 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3361 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3421 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3481 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3541 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3601 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3661 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3721 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3781 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3841 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3901 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3961 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4021 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4081 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4141 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4201 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4261 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4321 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4381 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4441 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4501 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4561 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4621 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4681 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4741 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4801 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4861 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4921 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4981 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5041 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     5101 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
