LOCUS       sequence-413789-        4317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MMEJ donor template for moxGFP-2A-Puro insertion into the C-terminus
            of the H2BC11 locus for nuclei visualization. To be co-transfected 
            with sgRNA plasmid px330-PITCh-H2BC11 Addgene #207755.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4317)
  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 4317)
  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-C-moxGFP-Puro-H2BC11-MMEJ
FEATURES             Location/Qualifiers
     source          1..4317
                     /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"
     CDS             join(474..476,477..479,480..1190)
                     /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(519..540)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1127..1148
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1200..1226
                     /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             1200..1226
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1230..1256
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1263..1296)
                     /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             1308..1364
                     /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"
     CDS             1371..1967
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1371..1390)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1867..1887
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1974..2007)
                     /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)."
     primer_bind     complement(2096..2112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2096..2112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2109..2131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2120..2136
                     /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(2144..2150,2151..2168,2169..2174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2189..2210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2327..2344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2498..3086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2578..2597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3257..4048,4049..4117))
                     /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     3880..3899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4118..4222)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4290..4308
                     /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 cgcatcgtac gcgtacgtgt
      421 ttggcgtcac caagtacacc agcgctggag gaagcggagg atctggcggt accatggtga
      481 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      541 tgaacggcca caagttctcc gtgcggggcg agggcgaggg cgatgccacc aacggcaagc
      601 tgaccctgaa gttcatcagc accaccggca agctgcccgt gccctggccc accctcgtga
      661 ccaccctgac ctacggcgtg cagagcttct cccgctaccc cgaccacatg aagcgccacg
      721 acttcttcaa gagcgccatg cccgaaggct acgtccagga gcgcaccatc tccttcaagg
      781 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
      841 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
      901 agtacaactt caactcccac aacgtctata tcaccgccga caagcagaag aacggcatca
      961 aggccaactt caagatccgc cataacgtgg aggacggctc cgtgcagctc gccgaccact
     1021 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctgt
     1081 ccacccagtc caagctgtcc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1141 agttcgtgac cgccgccggg atcactcacg gcatggacga gctgtacaag ggctctggct
     1201 acccctacga cgtgcccgac tacgccggct atccgtatga tgtcccggac tatgcaacgc
     1261 gttaccgttc gtatagcata cattatacga agttatctgg atccggagct actaacttca
     1321 gcctgctgaa gcaggccgga gacgtggagg agaaccctgg accttctaga atgaccgagt
     1381 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     1441 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     1501 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     1561 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     1621 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     1681 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     1741 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     1801 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     1861 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     1921 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccccc gggataactt
     1981 cgtatagcat acattatacg aacggtagcc tcgagaccgg taagagcaag tgagctaagt
     2041 aaacagtgag ttggccaaac acgtacgcgt acgatgcaag cttggcgtaa tcatggtcat
     2101 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2161 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2221 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2281 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2341 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2401 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2461 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2521 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2581 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2641 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2701 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2761 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2821 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2881 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2941 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3001 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3061 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3121 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3181 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3241 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3301 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3361 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3421 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3481 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3541 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3601 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3661 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3721 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3781 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3841 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3901 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3961 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4021 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4081 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4141 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4201 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4261 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
