LOCUS       sequence-451271-        4668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty C-terminus donor cassette. Integrate homology arms to target 
            the C-terminus insertion of a mScarlet-EFS-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4668)
  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 4668)
  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-EFS-Puro
FEATURES             Location/Qualifiers
     source          1..4668
                     /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             444..1139
                     /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(577..595)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     845..864
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1155..1196
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1218..1251)
                     /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)."
     polyA_signal    1373..1494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1410..1429)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1464..1483
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1544..1755
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1766..2362
                     /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(1766..1785)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2262..2282
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2369..2402)
                     /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(2447..2463)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2447..2463)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2460..2482)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2471..2487
                     /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(2495..2501,2502..2519,2520..2525))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2540..2561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2678..2695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2849..3437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2929..2948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3608..4399,4400..4468))
                     /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     4231..4250
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4469..4573)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4641..4659
                     /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 cggaggaagc ggaggatctg
      421 gcggtaccgg tggaggcgga tctatggtga gcaagggcga ggcagtgatc aaggagttca
      481 tgcggttcaa ggtgcacatg gagggctcca tgaacggcca cgagttcgag atcgagggcg
      541 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      601 gccccctgcc cttctcctgg gacatcctgt cccctcagtt catgtacggc tccagggcct
      661 tcaccaagca ccccgccgac atccccgact actataagca gtccttcccc gagggcttca
      721 agtgggagcg cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacct
      781 ccctggagga cggcaccctg atctacaagg tgaagctccg cggcaccaac ttccctcctg
      841 acggccccgt aatgcagaag aagacaatgg gctgggaagc gtccaccgag cggttgtacc
      901 ccgaggacgg cgtgctgaag ggcgacatta agatggccct gcgcctgaag gacggcggcc
      961 gctacctggc ggacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg
     1021 cctacaacgt cgaccgcaag ttggacatca cctcccacaa cgaggactac accgtggtgg
     1081 aacagtacga acgctccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     1141 gaggtggcgg ttccggcaag cccatcccca accccctgct gggcctggac agcaccggag
     1201 gtggcggatc aacgcgttac cgttcgtata gcatacatta tacgaagtta tctggatcct
     1261 gataatgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     1321 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     1381 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     1441 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttactagat
     1501 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     1561 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg
     1621 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     1681 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     1741 gccgccagaa cacagggcct ctagaatgac cgagtacaag cccacggtgc gcctcgccac
     1801 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     1861 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     1921 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     1981 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2041 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     2101 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     2161 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     2221 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     2281 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     2341 catgacccgc aagcccggtg cccccgggat aacttcgtat agcatacatt atacgaacgg
     2401 tagcctcgag accggtaaga gcaagtgaaa gcttggcgta atcatggtca tagctgtttc
     2461 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2521 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2581 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2641 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2701 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2761 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2821 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2881 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2941 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3001 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3061 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3121 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3181 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3241 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3301 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3361 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3421 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3481 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3541 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3601 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3661 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3721 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3781 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3841 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3901 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3961 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4021 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4081 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4141 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4201 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4261 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4321 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4381 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4441 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4501 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4561 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4621 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
