LOCUS       sequence-415319-        5101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for moxGFP-2A-Zeo 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 5101)
  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 5101)
  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-Zeo-H2BC11
FEATURES             Location/Qualifiers
     source          1..5101
                     /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..2246
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     protein_bind    complement(2253..2286)
                     /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(2880..2896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2880..2896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2893..2915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2904..2920
                     /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(2928..2934,2935..2952,2953..2958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2973..2994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3111..3128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3282..3870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3362..3381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4041..4832,4833..4901))
                     /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     4664..4683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4902..5006)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5074..5092
                     /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 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     1921 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt cagccgggac ttcgtggagg
     1981 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     2041 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     2101 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     2161 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     2221 tgcacttcgt ggccgaggag caggaccccg ggataacttc gtatagcata cattatacga
     2281 acggtagcct cgagaccggt aagagcaagt gataaacagt gagttggttg caaactctca
     2341 accctaacgg ctcttttaag agccacccat gttctcaaag aaagagctgg tgcttgtatt
     2401 cctcctctgc tggccactga caaacccttg taacttgcta ctgtgttttt tggtctgaag
     2461 tagagcagtt atttaactaa tccttagtga cttttttttt ttagatctgc cattctaatc
     2521 ttagagttaa gtaaggagat gggaaatttt ctattataag ttcgaaacca attaaaatac
     2581 gttagaaacc catcaaaata ctcgtcggtc ccccgtcggt tagtgatttg gaacagtgcc
     2641 aagttgcagc ggttgtcagt ttgaatttgc ccgggcaacg cccgcccttc ctctgcatcc
     2701 tgattggttg ttgtatccag gaagtccacg gtcggcttgg gtgcgccaaa ggaatccaaa
     2761 ccccgccttt atttggcctt ccttacaaac tcagtaaact gtcagtttct cattctttag
     2821 tattttatcg ctttcgggaa ggagatttat ttctaattag taagcttggc gtaatcatgg
     2881 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2941 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3001 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3061 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3121 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3181 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3241 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3301 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3361 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3421 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3481 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3541 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3601 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3661 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3721 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3781 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3841 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3901 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3961 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4021 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4081 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4141 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4201 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4261 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4321 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4381 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4441 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4501 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4561 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4621 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4681 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4741 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4801 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4861 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4921 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4981 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     5041 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     5101 c
//
