LOCUS       sequence-413780-        3556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty C-terminus donor cassette. Integrate homology arms to target 
            the C-terminus insertion of a 3xHA-2A-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3556)
  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 3556)
  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-3xHA-Puro
FEATURES             Location/Qualifiers
     source          1..3556
                     /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..524
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     protein_bind    complement(546..579)
                     /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             591..647
                     /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             654..1250
                     /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(654..673)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1150..1170
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1257..1290)
                     /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(1335..1351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1335..1351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1348..1370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1359..1375
                     /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(1383..1389,1390..1407,1408..1413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1428..1449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1566..1583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1737..2325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1817..1836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2496..3287,3288..3356))
                     /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     3119..3138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3357..3461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3529..3547
                     /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 tcttacccat acgatgttcc agattacgct tacccctacg
      481 acgtgcctga ttatgcctac ccatacgatg tgccagacta tgccggaggt ggcggatcaa
      541 cgcgttaccg ttcgtatagc atacattata cgaagttatc tggatccgga gctactaact
      601 tcagcctgct gaagcaggcc ggagacgtgg aggagaaccc tggaccttct agaatgaccg
      661 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
      721 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
      781 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
      841 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
      901 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
      961 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     1021 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     1081 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     1141 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     1201 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc cccgggataa
     1261 cttcgtatag catacattat acgaacggta gcctcgagac cggtaagagc aagtgaaagc
     1321 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1381 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1441 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1501 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1561 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1621 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1681 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1741 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1801 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1861 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1921 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1981 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2041 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2101 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2161 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2221 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2281 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2341 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2401 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2581 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3481 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3541 acgaggccct ttcgtc
//
