LOCUS       sequence-416185-        5326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for moxGFP-2A-Puro insertion into the C-terminus of 
            the H3C2 locus for nuclei visualization. To be co-transfected with 
            sgRNA plasmid px330-PITCh-H3C2 Addgene #207780.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5326)
  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 5326)
  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-H3C2
FEATURES             Location/Qualifiers
     source          1..5326
                     /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(978..980,981..983,984..1694)
                     /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(1023..1044)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1631..1652
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1704..1730
                     /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             1704..1730
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1734..1760
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1767..1800)
                     /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             1812..1868
                     /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             1875..2471
                     /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(1875..1894)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2371..2391
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2478..2511)
                     /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(3105..3121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3105..3121)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3118..3140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3129..3145
                     /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(3153..3159,3160..3177,3178..3183))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3198..3219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3336..3353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3507..4095)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3587..3606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4266..5057,5058..5126))
                     /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     4889..4908
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5127..5231)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5299..5317
                     /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 cgatggacca atccaagaag
      421 ggcgcgggga tttttgaatt ttcttgggtc caatagttgg tggtctgact ctataaaaga
      481 agagtagctc tttcctttcc tccacagacg tctctgcagg caagcttttc tgtggttttg
      541 ccatggctcg tactaaacag acagctcgga aatccaccgg cggtaaagcg ccacgcaagc
      601 agctggctac caaggctgct cgcaagagcg cgccggctac cggcggcgtg aaaaagcctc
      661 accgttaccg cccgggcact gtggctctgc gcgagatccg ccgctaccaa aagtcgaccg
      721 agttgctgat tcggaagctg ccgttccagc gcctggtgcg agaaatcgcc caagacttca
      781 agaccgatct tcgcttccag agctctgcgg tgatggcgct gcaggaggct tgtgaggcct
      841 acttggtagg gctctttgag gacacaaacc tttgcgccat ccatgctaag cgagtgacta
      901 ttatgcccaa agacatccag ctcgctcggc gcattcgcgg agaaagagcg ggaggaagcg
      961 gaggatctgg cggtaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1021 tcctggtcga gctggacggc gacgtgaacg gccacaagtt ctccgtgcgg ggcgagggcg
     1081 agggcgatgc caccaacggc aagctgaccc tgaagttcat cagcaccacc ggcaagctgc
     1141 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagagc ttctcccgct
     1201 accccgacca catgaagcgc cacgacttct tcaagagcgc catgcccgaa ggctacgtcc
     1261 aggagcgcac catctccttc aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt
     1321 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1381 gcaacatcct ggggcacaag ctggagtaca acttcaactc ccacaacgtc tatatcaccg
     1441 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccataac gtggaggacg
     1501 gctccgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1561 tgctgcccga caaccactac ctgtccaccc agtccaagct gtccaaagac cccaacgaga
     1621 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact cacggcatgg
     1681 acgagctgta caagggctct ggctacccct acgacgtgcc cgactacgcc ggctatccgt
     1741 atgatgtccc ggactatgca acgcgttacc gttcgtatag catacattat acgaagttat
     1801 ctggatccgg agctactaac ttcagcctgc tgaagcaggc cggagacgtg gaggagaacc
     1861 ctggaccttc tagaatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     1921 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     1981 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     2041 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     2101 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     2161 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     2221 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     2281 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     2341 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     2401 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     2461 agcccggtgc ccccgggata acttcgtata gcatacatta tacgaacggt agcctcgaga
     2521 ccggtaagag caagtgataa atgtaaagtc actttttcat cagtcttaaa acccaaagtc
     2581 tcttttcaga gccacccact tattccaacg aaagtagctg tgataatttt ttgttgtctt
     2641 aacagaacaa atttctaagg acccccccgg aaagcattag actatggtct taaagttgat
     2701 taacagaaat aacggtttgg tcagtcttgc agtgtaggtt atttctgacc ttattaaggt
     2761 gctatttgga gagaagctgt gtaagtccac tatcattcag gcctctagct tgctatgatt
     2821 agcatttgtt taaacaactt tgtaagagta agggaaaaat ctggtaagta gttaactggc
     2881 gcttactagg catttttgca aagctttgaa aagattagaa aattgtgtct tgcgagttcc
     2941 agtgtcttcc tcaaaatgct taggaagatt ttctcagctc aatacatagt cccctaggtt
     3001 ttctcatata ttatatctat atctacatat atatctactg ttaaattcat ttggctgtta
     3061 acattaacct gaaatttatt ctggtgaagc ttggcgtaat catggtcata gctgtttcct
     3121 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3181 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3241 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3301 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3361 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3421 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3481 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3541 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3601 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3661 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3721 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3781 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3841 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3901 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3961 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4021 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4081 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4141 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4201 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4261 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4321 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4381 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4441 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4501 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4561 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4621 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4681 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4741 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4801 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4861 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4921 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4981 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5041 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5101 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5161 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5221 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     5281 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
