LOCUS       sequence-228148-        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"
     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"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             603..977
                     /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    987..1211
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1218..1466
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1218..1238
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1389..1408
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1468..1501)
                     /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(1538..1557)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1539..1557)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1644..1661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1815..2403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1895..1914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2574..3365,3366..3434))
                     /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     3197..3216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3435..3539)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3607..3625
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3663..3685)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3785..3804
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3885..3901,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3885..3901,1)
                     /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 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
      661 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
      721 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
      781 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
      841 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
      901 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
      961 ccgaggagca ggactgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg
     1021 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1081 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1141 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1201 gggctctatg gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     1261 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     1321 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     1381 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     1441 atatcttgtg gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta
     1501 tggtaccgat gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt
     1561 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1621 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1681 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1741 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1801 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1861 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1921 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1981 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     2041 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2101 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2161 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2221 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     2281 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2341 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2401 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2461 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2521 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2581 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2641 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2701 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2761 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2821 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2881 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2941 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3001 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3061 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3121 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3181 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3241 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3301 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3361 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3421 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3481 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3541 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     3601 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3661 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3721 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3781 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa
     3841 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     3901 a
//
