LOCUS       sequence-413805-        4240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty C-terminus donor cassette. Integrate homology arms to target 
            the C-terminus insertion of a mNeon-2A-Puro cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4240)
  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 4240)
  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-mNeon-Puro
FEATURES             Location/Qualifiers
     source          1..4240
                     /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..1151
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      699..708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1167..1208
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1230..1263)
                     /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             1275..1331
                     /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             1338..1934
                     /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(1338..1357)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1834..1854
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1941..1974)
                     /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(2019..2035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2019..2035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2032..2054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2043..2059
                     /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(2067..2073,2074..2091,2092..2097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2112..2133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2250..2267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2421..3009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2501..2520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3180..3971,3972..4040))
                     /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     3803..3822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4041..4145)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4213..4231
                     /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 ggaggataac atggcctctc
      481 tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg
      541 tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca
      601 agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc
      661 agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg
      721 gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc
      781 gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc
      841 ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga
      901 agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa
      961 atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg
     1021 ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca
     1081 agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg
     1141 agctgtacaa gggaggtggc ggttccggca agcccatccc caaccccctg ctgggcctgg
     1201 acagcaccgg aggtggcgga tcaacgcgtt accgttcgta tagcatacat tatacgaagt
     1261 tatctggatc cggagctact aacttcagcc tgctgaagca ggccggagac gtggaggaga
     1321 accctggacc ttctagaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     1381 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1441 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1501 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1561 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1621 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1681 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1741 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1801 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1861 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1921 gcaagcccgg tgcccccggg ataacttcgt atagcataca ttatacgaac ggtagcctcg
     1981 agaccggtaa gagcaagtga aagcttggcg taatcatggt catagctgtt tcctgtgtga
     2041 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2101 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2161 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2221 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2281 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2341 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2401 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2461 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2521 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2581 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2641 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2701 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2761 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2821 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2881 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2941 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3001 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3061 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3121 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3181 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3241 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3301 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3361 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3421 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3481 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3541 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3601 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3661 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3721 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3781 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3841 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3901 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3961 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4021 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4081 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4141 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4201 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
