LOCUS       sequence-414347-        4228 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-2A-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4228)
  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 4228)
  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
FEATURES             Location/Qualifiers
     source          1..4228
                     /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
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGCEASTERLYPEDGVLKGDIK
                     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)."
     CDS             1263..1319
                     /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             1326..1922
                     /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(1326..1345)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1822..1842
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1929..1962)
                     /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(2007..2023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2007..2023)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2020..2042)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2031..2047
                     /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(2055..2061,2062..2079,2080..2085))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2100..2121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2238..2255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2409..2997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2489..2508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3168..3959,3960..4028))
                     /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     3791..3810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4029..4133)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4201..4219
                     /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 gctgtgaagc 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 tctggatccg
     1261 gagctactaa cttcagcctg ctgaagcagg ccggagacgt ggaggagaac cctggacctt
     1321 ctagaatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     1381 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     1441 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     1501 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     1561 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     1621 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     1681 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     1741 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     1801 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     1861 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     1921 cccccgggat aacttcgtat agcatacatt atacgaacgg tagcctcgag accggtaaga
     1981 gcaagtgaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2041 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2101 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2161 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2221 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2281 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2341 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2401 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2461 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2521 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2581 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2641 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2701 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2761 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2821 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2881 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2941 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3001 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3061 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3121 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3181 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3241 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3301 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3361 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3421 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3481 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3541 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3601 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3661 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3721 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3781 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3841 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3901 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3961 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4021 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4081 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4141 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4201 aataggcgta tcacgaggcc ctttcgtc
//
