LOCUS       sequence-232647-        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"
     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"
     polyA_signal    1229..1453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1460..1708
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1460..1480
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1631..1650
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1710..1743)
                     /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(1780..1799)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1781..1799)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1886..1903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2057..2645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2137..2156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2816..3607,3608..3676))
                     /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     3439..3458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3677..3781)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3849..3867
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3905..3927)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4027..4046
                     /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 tgactgagta caagcctact gtgcgtttag ccaccaggga
      661 tgatgtgcca cgagccgtga ggacactagc agccgccttt gctgactacc cagctacaag
      721 gcacaccgtg gaccctgaca gacacattga aagagtcaca gaacttcagg agctgttttt
      781 aactagggtg ggcctggata ttggcaaagt ctgggtggcc gatgatggag cagcagttgc
      841 agtgtggacc accccagagt ctgttgaagc tggagctgtc tttgcagaaa ttggaccaag
      901 aatggccgag ttatctggca gcaggctggc tgctcaacag cagatggaag gattattagc
      961 tccccacaga cccaaggagc ctgcctggtt cctggcaaca gttggagttt ctccagacca
     1021 tcagggcaag ggcttaggat ctgctgtggt gctgcctggt gtggaagcag ctgagagagc
     1081 tggggtgcca gcatttctgg agacatcagc tcctagaaac ctgcccttct atgaacggct
     1141 gggcttcaca gtgactgctg atgtggaggt gcctgagggc cccagaacct ggtgcatgac
     1201 aagaaaacca ggagcctgac tgcagcgact gtgccttcta gttgccagcc atctgttgtt
     1261 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1321 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1381 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1441 gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     1501 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     1561 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     1621 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     1681 atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt
     1741 tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa
     1801 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1861 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1921 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1981 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2041 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2101 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2161 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2221 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2281 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2341 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2401 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2461 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2521 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2581 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2641 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2701 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2761 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2821 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2881 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2941 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3001 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3061 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3121 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3181 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3241 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3301 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3361 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3421 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3481 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3541 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3601 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3661 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3721 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3781 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     3841 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     3901 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     3961 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4021 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     4081 aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
