LOCUS       sequence-413807-        3856 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Puro-2A-dTAG cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3856)
  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 3856)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Puro-dTAG
FEATURES             Location/Qualifiers
     source          1..3856
                     /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..1061
                     /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(465..484)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     961..981
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1071..1127
                     /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(1134..1167)
                     /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             1191..1232
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1248..1568
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     primer_bind     complement(1635..1651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1635..1651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1648..1670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1659..1675
                     /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(1683..1689,1690..1707,1708..1713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1728..1749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1866..1883)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2037..2625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2117..2136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2796..3587,3588..3656))
                     /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     3419..3438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3657..3761)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3829..3847
                     /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 tagcatgacc gagtacaagc
      481 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
      541 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
      601 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
      661 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
      721 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
      781 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
      841 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
      901 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
      961 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     1021 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatccgga gctactaact
     1081 tcagcctgct gaagcaggcc ggagacgtgg aggagaaccc tggaccttct agaataactt
     1141 cgtatagcat acattatacg aacggtagca cgcgtggagg tggcggttcc ggcaagccca
     1201 tccccaaccc cctgctgggc ctggacagca ccggaggtgg cggatcagga gtgcaggtgg
     1261 aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc
     1321 actacaccgg gatgcttgaa gatggaaaga aagttgattc cagccgggac agaaacaagc
     1381 cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc
     1441 agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca
     1501 ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa
     1561 aactggaagg tggaggcgga tctctcgagg gaggaagcgg aggatctggc ggtaccaagc
     1621 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1681 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1741 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1861 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1921 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1981 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2041 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2101 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2161 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2221 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2281 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2341 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2401 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2461 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2521 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2581 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2641 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2701 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2761 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2821 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2881 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2941 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3001 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3061 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3121 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3181 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3241 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3301 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3361 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3421 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3481 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3541 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3601 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3661 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3721 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3781 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3841 acgaggccct ttcgtc
//
