LOCUS       sequence-228078-        2957 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with VSV-G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2957)
  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 2957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-30
FEATURES             Location/Qualifiers
     source          1..2957
                     /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..522
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     274..294
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     445..464
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(524..557)
                     /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(594..613)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(595..613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(700..717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(871..1459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(951..970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1630..2421,2422..2490))
                     /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     2253..2272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2491..2595)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2663..2681
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2719..2741)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2841..2860
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2941..2957,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2941..2957,1)
                     /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 gaggagggcc tatttcccat gattccttca
      301 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      361 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      421 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      481 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat
      541 acattatacg aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata
      601 gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc
      661 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
      721 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      781 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      841 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      901 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      961 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1021 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     1081 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1141 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1201 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1261 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     1321 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1381 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1441 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1501 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1561 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1621 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1681 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1741 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1801 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1861 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1921 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1981 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2041 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2101 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2161 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2221 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2281 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2341 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2401 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2461 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2521 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2581 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     2641 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     2701 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2761 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2821 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga
     2881 catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     2941 atttaggtga cactata
//
