LOCUS       sequence-415158-        5125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for moxGFP-2A-Blast 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 5125)
  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 5125)
  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-Blast-H2BC11
FEATURES             Location/Qualifiers
     source          1..5125
                     /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             573..950
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     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..2270
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(2277..2310)
                     /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(2904..2920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2904..2920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2917..2939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2928..2944
                     /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(2952..2958,2959..2976,2977..2982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2997..3018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3135..3152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3306..3894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3386..3405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4065..4856,4857..4925))
                     /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     4688..4707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4926..5030)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5098..5116
                     /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 catcctgatt gggcagacct
      421 gacctctgac gttaccctga ataactacca atcagacaca agacttcaac tcttcacctt
      481 atttgcataa gcgattctat ataaaagcgc cttgtcatac cctgctcacg ctgtttttcc
      541 ttttcgttgg cgctttatag ctacacagtg ctatgccaga gccagcgaag tctgctcccg
      601 ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaagac ggcaagaagc
      661 gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg aagcaggtcc
      721 accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt gtgaacgaca
      781 ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag cgctcgacca
      841 tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag ttggccaagc
      901 acgccgtgtc cgagggtact aaggccgtca ccaagtacac tagcgctaag 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 tagaatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     1921 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     1981 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     2041 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     2101 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     2161 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     2221 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc cccgggataa
     2281 cttcgtatag catacattat acgaacggta gcctcgagac cggtaagagc aagtgataaa
     2341 cagtgagttg gttgcaaact ctcaacccta acggctcttt taagagccac ccatgttctc
     2401 aaagaaagag ctggtgcttg tattcctcct ctgctggcca ctgacaaacc cttgtaactt
     2461 gctactgtgt tttttggtct gaagtagagc agttatttaa ctaatcctta gtgacttttt
     2521 ttttttagat ctgccattct aatcttagag ttaagtaagg agatgggaaa ttttctatta
     2581 taagttcgaa accaattaaa atacgttaga aacccatcaa aatactcgtc ggtcccccgt
     2641 cggttagtga tttggaacag tgccaagttg cagcggttgt cagtttgaat ttgcccgggc
     2701 aacgcccgcc cttcctctgc atcctgattg gttgttgtat ccaggaagtc cacggtcggc
     2761 ttgggtgcgc caaaggaatc caaaccccgc ctttatttgg ccttccttac aaactcagta
     2821 aactgtcagt ttctcattct ttagtatttt atcgctttcg ggaaggagat ttatttctaa
     2881 ttagtaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2941 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3001 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3061 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3121 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3301 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3901 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3961 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4021 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4081 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4141 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4201 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4261 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4321 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4381 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4441 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4501 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4561 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4621 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4681 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4741 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4801 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4861 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4921 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4981 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5041 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5101 aggcgtatca cgaggccctt tcgtc
//
