LOCUS       sequence-416864-        5800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-mNeon 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 5800)
  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 5800)
  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-mNeon-ACTB
FEATURES             Location/Qualifiers
     source          1..5800
                     /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..2726
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2274..2283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3579..3595)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3579..3595)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3592..3614)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3603..3619
                     /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(3627..3633,3634..3651,3652..3657))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3672..3693
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3810..3827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3981..4569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4061..4080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4740..5531,5532..5600))
                     /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     5363..5382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5601..5705)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5773..5791
                     /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 ggcgaggagg
     2041 ataacatggc ctctctccca gcgacacatg agttacacat ctttggctcc atcaacggtg
     2101 tggactttga catggtgggt cagggcaccg gcaatccaaa tgatggttat gaggagttaa
     2161 acctgaagtc caccaagggt gacctccagt tctccccctg gattctggtc cctcatatcg
     2221 ggtatggctt ccatcagtac ctgccctacc ctgacgggat gtcgcctttc caggccgcca
     2281 tggtagatgg ctccggatac caagtccatc gcacaatgca gtttgaagat ggtgcctccc
     2341 ttactgttaa ctaccgctac acctacgagg gaagccacat caaaggagag gcccaggtga
     2401 aggggactgg tttccctgct gacggtcctg tgatgaccaa ctcgctgacc gctgcggact
     2461 ggtgcaggtc gaagaagact taccccaacg acaaaaccat catcagtacc tttaagtgga
     2521 gttacaccac tggaaatggc aagcgctacc ggagcactgc gcggaccacc tacacctttg
     2581 ccaagccaat ggcggctaac tatctgaaga accagccgat gtacgtgttc cgtaagacgg
     2641 agctcaagca ctccaagacc gagctcaact tcaaggagtg gcaaaaggcc tttaccgatg
     2701 tgatgggcat ggacgagctg tacaagctcg agggaggaag cggaggatct ggcggtaccg
     2761 atgatgatat cgcagcgctc gtcgtcgaca acggctccgg catgtgcaag gccggcttcg
     2821 cgggcgacga tgccccccgg gccgtcttcc cctccatcgt ggggcgcccc aggcaccagg
     2881 taggggagct ggctgggtgg ggcagccccg ggagcgggcg ggaggcaagg gcgctttctc
     2941 tgcacaggag cctcccggtt tccggggtgg gggctgcgcc cgtgctcagg gcttcttgtc
     3001 ctttccttcc cagggcgtga tggtgggcat gggtcagaag gattcctatg tgggcgacga
     3061 ggcccagagc aagagaggca tcctcaccct gaagtacccc atcgagcacg gcatcgtcac
     3121 caactgggac gacatggaga aaatctggca ccacaccttc tacaatgagc tgcgtgtggc
     3181 tcccgaggag caccccgtgc tgctgaccga ggcccccctg aaccccaagg ccaaccgcga
     3241 gaagatgacc caggtgagtg gcccgctacc tcttctggtg gccgcctccc tccttcctgg
     3301 cctcccggag ctgcgccctt tctcactggt tctctcttct gccgttttcc gtaggactct
     3361 cttctctgac ctgagtctcc tttggaactc tgcaggttct atttgctttt tcccagatga
     3421 gctctttttc tggtgtttgt ctctctgact aggtgtctaa gacagtgttg tgggtgtagg
     3481 tactaacact ggctcgtgtg acaaggccat gaggctggtg taaagcggcc ttggagtgtg
     3541 tattaagtag gcgcacagta aagcttggcg taatcatggt catagctgtt tcctgtgtga
     3601 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3661 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3721 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3781 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3841 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3901 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3961 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4021 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4081 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4141 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4201 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4261 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4321 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4381 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4441 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4501 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4561 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4621 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4681 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4741 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4801 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4861 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4921 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4981 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5041 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5101 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5161 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5221 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5281 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5341 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5401 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5461 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5521 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5581 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5641 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5701 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5761 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
