LOCUS       sequence-232671-        3934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3934)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 3934)
  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: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-Z34
FEATURES             Location/Qualifiers
     source          1..3934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..146
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    156..277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(193..212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     247..266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(284..317)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        324..640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      491..626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      647..656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             653..1027
                     /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"
     polyA_signal    1037..1261
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1268..1516
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1268..1288
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1439..1458
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1518..1551)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1588..1607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1589..1607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1694..1711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1865..2453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1945..1964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2624..3415,3416..3484))
                     /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     3247..3266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3485..3589)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3657..3675
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3713..3735)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3835..3854
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gactacaaag accatgacgg tgattataaa gatcatgata
      121 tcgattacaa ggatgacgat gacaagtaga ctagtaactt gtttattgca gcttataatg
      181 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      241 ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt cgtatagcat
      301 acattatacg aagttatgtc gacggtgtgg aaagtcccca ggctccccag caggcagaag
      361 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
      421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
      481 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
      541 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
      601 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca ccatggccaa
      661 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
      721 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
      781 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
      841 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
      901 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
      961 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     1021 ggactgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1081 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1141 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1201 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     1261 gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     1321 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     1381 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     1441 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     1501 gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta tggtaccgat
     1561 gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt tcgataagcc
     1621 aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1681 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1741 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1801 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2701 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2761 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2821 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2881 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2941 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3001 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3061 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3121 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3181 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3241 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3301 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3361 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3601 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     3661 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3721 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3781 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     3841 cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa cgcggctaca
     3901 attaatacat aaccttatgt atcatacaca tacg
//
