LOCUS       sequence-413803-        5152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-sTagRFP 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 5152)
  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 5152)
  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-sTagRFP-TUBA1B
FEATURES             Location/Qualifiers
     source          1..5152
                     /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             1599..2330
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTV
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(2931..2947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2931..2947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2944..2966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2955..2971
                     /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(2979..2985,2986..3003,3004..3009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3024..3045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3162..3179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3333..3921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3413..3432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4092..4883,4884..4952))
                     /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     4715..4734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4953..5057)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5125..5143
                     /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 ggtgtctaag ggcgaggaac
     1621 tgattaagga gaatatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact
     1681 tcaaatgcac ctccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca
     1741 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt
     1801 acggcagcag aaccttcatc aaccacaccc agggcatccc cgatttcttt aagcagtcct
     1861 tcccagaggg cttcacatgg gagagagtca ccacatacga ggacgggggc gtgctgaccg
     1921 ccacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg
     1981 tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccaaca
     2041 ccgagatgct gtaccccgct gacggcggcc tggaaggcag aaccgtcatg gccctgaagc
     2101 tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacaggtcc aagaaacccg
     2161 ctaagaacct gaagatgccc ggagtgtact atgtggacca cagactggag agaatcaagg
     2221 aggccgacaa agagacatac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc
     2281 tccctagcaa actgggccac aagctgaacg gcatggacga gctgtacaag ctcgagggag
     2341 gaagcggagg atctggcggt accgagtgca tctccatcca cgttggccag gctggtgtcc
     2401 agattggcaa tgcctgctgg gagctctact gcctggaaca cggcatccag cccgatggcc
     2461 agatgccaag tgacaagacc attgggggag gagatgactc cttcaacacc ttcttcagtg
     2521 agacgggcgc tggcaagcac gtgccccggg ctgtgtttgt agacttggaa cccacagtca
     2581 ttggtgagtt gacctcagta acctgagatc ccaggatgct gggacaggag gtctgtccag
     2641 gggcttctct tgtcactcac tcactccctc cgtccttctc tccctcctcc agatgaagtt
     2701 cgcactggca cctaccgcca gctcttccac cctgagcagc tcatcacagg caaggaagat
     2761 gctgccaata actatgcccg agggcactac accattggca aggagatcat tgaccttgtg
     2821 ttggaccgaa ttcgcaagct ggtaagcacc acatataaat atgcatttaa tgtggtgtga
     2881 tagttccagt gcaagttggg tggagtgact gaaagcttgg cgtaatcatg gtcatagctg
     2941 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3001 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3061 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3121 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3181 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3241 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3301 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3361 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3481 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3541 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3661 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3721 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3781 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3841 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3901 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3961 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4021 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4081 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4141 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4201 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4261 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4321 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4381 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4441 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4501 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4561 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4621 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4681 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4741 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4801 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4861 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4921 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4981 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5041 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     5101 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
