LOCUS       sequence-232683-        3901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with VSV-G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3901)
  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 3901)
  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-Z30
FEATURES             Location/Qualifiers
     source          1..3901
                     /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..113
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     polyA_signal    123..244
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(160..179)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     214..233
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(251..284)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        291..607
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      458..593
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             620..994
                     /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    1004..1228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1235..1483
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1235..1255
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1406..1425
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1485..1518)
                     /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(1555..1574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1556..1574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1661..1678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1832..2420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1912..1931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2591..3382,3383..3451))
                     /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     3214..3233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3452..3556)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3624..3642
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3680..3702)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3802..3821
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tatacagaca tagagatgaa ccgacttgga aagtgaacta
      121 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      181 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      241 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgac ggtgtggaaa
      301 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
      361 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      421 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
      481 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
      541 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
      601 ttgcaaagaa ttcgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
      661 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
      721 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
      781 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
      841 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
      901 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
      961 ctgcgtgcac ttcgtggccg aggagcagga ctgactgcag cgactgtgcc ttctagttgc
     1021 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1081 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1141 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1201 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc
     1261 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     1321 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     1381 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     1441 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca
     1501 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat
     1561 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     1621 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1681 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1741 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1801 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1861 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1921 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1981 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2041 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2101 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2161 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2221 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2281 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2341 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2401 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2461 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2521 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2581 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2641 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2701 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2761 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2821 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2881 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2941 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3001 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3061 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3121 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3181 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3241 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3301 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3361 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3421 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3481 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3541 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     3601 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     3661 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3721 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3781 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     3841 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     3901 g
//
