LOCUS       sequence-416363-        5788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-mScarlet insertion into the N-terminus 
            of the ACTB locus for actin visualization. To be co-transfected with
            sgRNA plasmid px330-PITCh-ACTB Addgene #207748.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5788)
  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 5788)
  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-ACTB
FEATURES             Location/Qualifiers
     source          1..5788
                     /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(1401..1434)
                     /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             1443..1838
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1848..1904
                     /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(1911..1944)
                     /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             1953..1979
                     /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             1953..1979
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1983..2009
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2019..2714
                     /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(2152..2170)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2420..2439
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3567..3583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3567..3583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3580..3602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3591..3607
                     /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(3615..3621,3622..3639,3640..3645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3660..3681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3798..3815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3969..4557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4049..4068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4728..5519,5520..5588))
                     /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     5351..5370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5589..5693)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5761..5779
                     /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 ccctataaaa cccagcggcg
      421 cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag cacagagcct cgcctttgcc
      481 gatccgccgc ccgtccacac ccgccgccag gtaagcccgg ccagccgacc ggggcaggcg
      541 gctcacggcc cggccgcagg cggccgcggc cccttcgccc gtgcagagcc gccgtctggg
      601 ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt ggggggcgcg ggagaagccc
      661 ctgggcctcc ggagatgggg gacaccccac gccagttcgg aggcgcgagg ccgcgctcgg
      721 gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa ccggcggggt ctttgtctga
      781 gccgggctct tgccaatggg gatcgcaggg tgggcgcggc ggagcccccg ccaggcccgg
      841 tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt gggcgctaac tgcgtgcgcg
      901 ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca aggcgctaac tgcgcgtgcg
      961 ttctggggcc cggggtgccg cggcctgggc tggggcgaag gcgggctcgg ccggaagggg
     1021 tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct gcccgagccg ctggccgccc
     1081 gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc tgtttgaacc gggcggaggc
     1141 ggggctggcg cccggttggg agggggttgg ggcctggctt cctgccgcgc gccgcgggga
     1201 cgcctccgac cagtgtttgc cttttatggt aataacgcgg ccggcccggc ttcctttgtc
     1261 cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag gactcggcgc gccggaagtg
     1321 gccagggcgg gggcgacctc ggctcacagc gcgcccggct attctcgcaa ctgacaatgg
     1381 gcaagagcaa gaccggtagc taccgttcgt atagcataca ttatacgaag ttatctgcta
     1441 gcatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     1501 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     1561 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     1621 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     1681 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     1741 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     1801 ttcgtgaatt gctgccctct ggttatgtgt gggagggcgg atccggagct actaacttca
     1861 gcctgctgaa gcaggccgga gacgtggagg agaaccctgg accttctaga ataacttcgt
     1921 atagcataca ttatacgaac ggtagcacgc gttaccccta cgacgtgccc gactacgccg
     1981 gctatccgta tgatgtcccg gactatgcag gaagcggaat ggtgagcaag ggcgaggcag
     2041 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
     2101 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2161 aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
     2221 acggctccag ggccttcacc aagcaccccg ccgacatccc cgactactat aagcagtcct
     2281 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg
     2341 tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     2401 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca
     2461 ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc
     2521 tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg
     2581 tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg
     2641 actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg
     2701 acgagctgta caagctcgag ggaggaagcg gaggatctgg cggtaccgat gatgatatcg
     2761 cagcgctcgt cgtcgacaac ggctccggca tgtgcaaggc cggcttcgcg ggcgacgatg
     2821 ccccccgggc cgtcttcccc tccatcgtgg ggcgccccag gcaccaggta ggggagctgg
     2881 ctgggtgggg cagccccggg agcgggcggg aggcaagggc gctttctctg cacaggagcc
     2941 tcccggtttc cggggtgggg gctgcgcccg tgctcagggc ttcttgtcct ttccttccca
     3001 gggcgtgatg gtgggcatgg gtcagaagga ttcctatgtg ggcgacgagg cccagagcaa
     3061 gagaggcatc ctcaccctga agtaccccat cgagcacggc atcgtcacca actgggacga
     3121 catggagaaa atctggcacc acaccttcta caatgagctg cgtgtggctc ccgaggagca
     3181 ccccgtgctg ctgaccgagg cccccctgaa ccccaaggcc aaccgcgaga agatgaccca
     3241 ggtgagtggc ccgctacctc ttctggtggc cgcctccctc cttcctggcc tcccggagct
     3301 gcgccctttc tcactggttc tctcttctgc cgttttccgt aggactctct tctctgacct
     3361 gagtctcctt tggaactctg caggttctat ttgctttttc ccagatgagc tctttttctg
     3421 gtgtttgtct ctctgactag gtgtctaaga cagtgttgtg ggtgtaggta ctaacactgg
     3481 ctcgtgtgac aaggccatga ggctggtgta aagcggcctt ggagtgtgta ttaagtaggc
     3541 gcacagtaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3601 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3661 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3721 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3781 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3961 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4021 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4141 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4381 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5041 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5101 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5701 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     5761 aataggcgta tcacgaggcc ctttcgtc
//
