LOCUS       sequence-228079-        4126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with VSV-G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4126)
  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 4126)
  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-P30
FEATURES             Location/Qualifiers
     source          1..4126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..96
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     polyA_signal    106..227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(143..162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     197..216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(234..267)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        274..590
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      441..576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1212..1436
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1443..1691
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1443..1463
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1614..1633
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1693..1726)
                     /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(1763..1782)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1764..1782)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1869..1886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2040..2628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2120..2139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2799..3590,3591..3659))
                     /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     3422..3441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3660..3764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3832..3850
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3888..3910)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4010..4029
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4110..4126,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4110..4126,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctatacag acatagagat gaaccgactt ggaaagtgaa ctagtaactt gtttattgca
      121 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
      181 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt
      241 cgtatagcat acattatacg aagttatgtc gacggtgtgg aaagtcccca ggctccccag
      301 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
      361 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
      421 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
      481 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
      541 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca
      601 ccatgactga gtacaagcct actgtgcgtt tagccaccag ggatgatgtg ccacgagccg
      661 tgaggacact agcagccgcc tttgctgact acccagctac aaggcacacc gtggaccctg
      721 acagacacat tgaaagagtc acagaacttc aggagctgtt tttaactagg gtgggcctgg
      781 atattggcaa agtctgggtg gccgatgatg gagcagcagt tgcagtgtgg accaccccag
      841 agtctgttga agctggagct gtctttgcag aaattggacc aagaatggcc gagttatctg
      901 gcagcaggct ggctgctcaa cagcagatgg aaggattatt agctccccac agacccaagg
      961 agcctgcctg gttcctggca acagttggag tttctccaga ccatcagggc aagggcttag
     1021 gatctgctgt ggtgctgcct ggtgtggaag cagctgagag agctggggtg ccagcatttc
     1081 tggagacatc agctcctaga aacctgccct tctatgaacg gctgggcttc acagtgactg
     1141 ctgatgtgga ggtgcctgag ggccccagaa cctggtgcat gacaagaaaa ccaggagcct
     1201 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1261 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1321 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1381 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg
     1441 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     1501 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     1561 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     1621 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     1681 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     1741 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     1801 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1861 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1921 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1981 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2041 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2101 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2161 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2221 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2281 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2341 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2401 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2461 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2521 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2581 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2641 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2701 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2761 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2821 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2881 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2941 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3001 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3061 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3541 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3601 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3661 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3721 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3781 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3841 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3901 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3961 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4021 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     4081 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
