LOCUS       sequence-415325-        5305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mScarlet-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 5305)
  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 5305)
  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-mScarlet-Puro-H2BC11
FEATURES             Location/Qualifiers
     source          1..5305
                     /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             978..1673
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1111..1129)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1379..1398
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1683..1709
                     /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             1683..1709
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1713..1739
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1746..1779)
                     /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             1791..1847
                     /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             1854..2450
                     /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(1854..1873)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2350..2370
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2457..2490)
                     /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(3084..3100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3084..3100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3097..3119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3108..3124
                     /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(3132..3138,3139..3156,3157..3162))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3177..3198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3315..3332)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3486..4074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3566..3585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4245..5036,5037..5105))
                     /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     4868..4887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5106..5210)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5278..5296
                     /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 gcgaggcagt gatcaaggag ttcatgcggt
     1021 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
     1081 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1141 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcacca
     1201 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg
     1261 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg
     1321 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc
     1381 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg
     1441 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc
     1501 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca
     1561 acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt
     1621 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagggctctg
     1681 gctaccccta cgacgtgccc gactacgccg gctatccgta tgatgtcccg gactatgcaa
     1741 cgcgttaccg ttcgtatagc atacattata cgaagttatc tggatccgga gctactaact
     1801 tcagcctgct gaagcaggcc ggagacgtgg aggagaaccc tggaccttct agaatgaccg
     1861 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     1921 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     1981 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2041 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     2101 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2161 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     2221 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     2281 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     2341 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     2401 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc cccgggataa
     2461 cttcgtatag catacattat acgaacggta gcctcgagac cggtaagagc aagtgataaa
     2521 cagtgagttg gttgcaaact ctcaacccta acggctcttt taagagccac ccatgttctc
     2581 aaagaaagag ctggtgcttg tattcctcct ctgctggcca ctgacaaacc cttgtaactt
     2641 gctactgtgt tttttggtct gaagtagagc agttatttaa ctaatcctta gtgacttttt
     2701 ttttttagat ctgccattct aatcttagag ttaagtaagg agatgggaaa ttttctatta
     2761 taagttcgaa accaattaaa atacgttaga aacccatcaa aatactcgtc ggtcccccgt
     2821 cggttagtga tttggaacag tgccaagttg cagcggttgt cagtttgaat ttgcccgggc
     2881 aacgcccgcc cttcctctgc atcctgattg gttgttgtat ccaggaagtc cacggtcggc
     2941 ttgggtgcgc caaaggaatc caaaccccgc ctttatttgg ccttccttac aaactcagta
     3001 aactgtcagt ttctcattct ttagtatttt atcgctttcg ggaaggagat ttatttctaa
     3061 ttagtaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3121 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3181 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3241 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3301 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3361 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3421 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3481 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3541 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3601 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3661 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3721 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3781 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3841 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3901 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3961 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4021 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4081 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4141 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4201 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4261 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4321 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4381 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4441 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4501 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4561 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4621 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4681 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4741 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4801 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4861 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4921 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4981 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5041 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5101 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5221 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5281 aggcgtatca cgaggccctt tcgtc
//
