LOCUS       sequence-451310-        4045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty C-terminus donor cassette. Integrate homology arms to target 
            the C-terminus insertion of a ultraID-2A-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4045)
  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 4045)
  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-ultraID-Puro
FEATURES             Location/Qualifiers
     source          1..4045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..201
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     193..210
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     194..210
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             787..828
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(850..883)
                     /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             895..951
                     /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             958..1554
                     /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(958..977)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1454..1474
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1561..1594)
                     /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(1639..1655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1639..1655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1652..1674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1663..1679
                     /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(1687..1693,1694..1711,1712..1717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1732..1753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1870..1887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2041..2629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2121..2140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2800..3591,3592..3660))
                     /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     3423..3442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3661..3765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3833..3851
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3889..3911)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4011..4030
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg
       61 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
      121 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
      181 cagtcacgac gttgtaaaac gacggccagt gaattcggag gaagcggagg atctggcggt
      241 accggtggag gcggatctat gttcaagaac ctgatctggc tgaaggaggt ggacagcacc
      301 caggagagac tgaaggagtg gaacgtgtcc tacggcaccg ccctggtggc cgacagacag
      361 accaagggca gaggcggccc cggcagaaag tggctgagcc aggagggcgg cctgtacttc
      421 agcttcctgc tgaaccccaa ggagttcgag aacctgctgc agctgcccct ggtgctgggc
      481 ctgagcgtga gcgaggccct ggaggagatc accgagatcc ccttcagcct gaagtggccc
      541 aacgacgtgt acttccagga gaagaaggtg agcggcgtgc tgtgcgagct gagcaaggac
      601 aagctgatcg tgggcatcgg catcaacgtg aaccagagag agatccccga ggagatcaag
      661 gacagagcca ccaccctgta cgagatcacc ggcaaggact gggacagaaa ggaggtgctg
      721 ctgaaggtgc tgaagagaat cagcgagaac ctgaagaagt tcaaggagaa gggaggtggc
      781 ggttccggca agcccatccc caaccccctg ctgggcctgg acagcaccgg aggtggcgga
      841 tcaacgcgtt accgttcgta tagcatacat tatacgaagt tatctggatc cggagctact
      901 aacttcagcc tgctgaagca ggccggagac gtggaggaga accctggacc ttctagaatg
      961 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1021 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1081 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1141 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1201 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1261 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1321 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     1381 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     1441 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     1501 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcccccggg
     1561 ataacttcgt atagcataca ttatacgaac ggtagcctcg agaccggtaa gagcaagtga
     1621 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1681 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1741 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1801 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1861 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1921 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1981 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2041 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2101 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2161 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2221 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2281 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2341 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2401 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2461 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2521 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2581 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2641 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2701 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2761 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2821 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2881 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2941 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3001 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3061 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3121 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3181 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3241 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3301 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3361 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3421 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3481 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3541 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3601 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3661 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3721 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3781 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3841 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3901 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3961 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     4021 gcagattgta ctgagagtgc accat
//
