LOCUS       sequence-451979-        4018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Zeo-2A-mStayGold cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4018)
  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 4018)
  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-N-Zeo-mStayGold
FEATURES             Location/Qualifiers
     source          1..4018
                     /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"
     protein_bind    complement(423..456)
                     /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             465..836
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     CDS             846..902
                     /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"
     protein_bind    complement(909..942)
                     /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)."
     CDS             966..1007
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1023..1700
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     primer_bind     complement(1797..1813)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1797..1813)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1810..1832)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1821..1837
                     /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(1845..1851,1852..1869,1870..1875))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1890..1911
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2028..2045)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2199..2787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2279..2298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2958..3749,3750..3818))
                     /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     3581..3600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3819..3923)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3991..4009
                     /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 cggcaagagc aagaccggta
      421 gctaccgttc gtatagcata cattatacga agttatctgc tagcatggcc aagttgacca
      481 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
      541 ggctcgggtt cagccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
      601 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
      661 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
      721 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
      781 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggacggat
      841 ccggagctac taacttcagc ctgctgaagc aggccggaga cgtggaggag aaccctggac
      901 cttctagaat aacttcgtat agcatacatt atacgaacgg tagcacgcgt ggaggtggcg
      961 gttccggcaa gcccatcccc aaccccctgc tgggcctgga cagcaccgga ggtggcggaa
     1021 gcatggtgtc tacaggcgag gaactgttta ccggcgtggt gcccttcaag ttccagctga
     1081 agggcaccat caacggcaag agcttcaccg tggaaggcga gggcgagggc aatagccacg
     1141 agggcagcca caaaggcaag tatgtgtgca ccagcggcaa actgccaatg tcttgggccg
     1201 ccctgggaac tagcttcggc tatggcatga aatactatac caagtacccc agcggcctga
     1261 aaaactggtt ccacgaggtg atgcctgagg gcttcaccta cgacagacac atccagtaca
     1321 agggcgacgg cagcatccac gccaagcacc agcacttcat gaagaacggc acctaccaca
     1381 acatcgtgga gttcaccggc caggacttca aggagaacag ccccgtgctg accggcgaca
     1441 tggacgtgag cctgcccaac gaggtgcagc acatccccat tgatgacggc gtggagtgca
     1501 cagtgaccct gcagtaccct ctgctgagcg acgaaagcaa gtgcgtggaa gcctatcaga
     1561 acaccatcat caagcccctg cacaatcagc cagcccccga tgtgccattt cactggatca
     1621 gaaagcagta cacccagagc aaggacgaca ccgaggagag agaccacatc atccagagcg
     1681 agaccctgga ggcccacctg ccttggcatg agcctagcgc aagcgcagtg ggaggtggcg
     1741 gttccctcga gggaggaagc ggaggatctg gcggtaccaa gcttggcgta atcatggtca
     1801 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1861 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1921 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1981 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2041 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2101 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2161 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2221 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2281 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2341 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2401 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2461 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2521 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2581 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2641 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2701 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2761 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2821 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2881 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2941 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3001 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3061 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3121 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3181 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3241 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3301 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3361 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3421 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3481 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3541 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3601 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3661 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3721 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3781 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3841 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3901 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     3961 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
