LOCUS       sequence-232704-        4579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4579)
  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 4579)
  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-Z06
FEATURES             Location/Qualifiers
     source          1..4579
                     /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"
     polyA_signal    801..922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(838..857)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     892..911
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(929..962)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        969..1285
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1136..1271
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1292..1301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1298..1672
                     /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    1682..1906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1913..2161
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1913..1933
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2084..2103
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2163..2196)
                     /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(2233..2252)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2234..2252)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2339..2356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2510..3098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2590..2609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3269..4060,4061..4129))
                     /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     3892..3911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4130..4234)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4302..4320
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4358..4380)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4480..4499
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc agcaagggcg aggagctgtt caccggcgtg gtgcccatcc
      121 tggtggagct ggacggcgac gtgaacggcc acaagttcag cgtgcgcggc gagggcgagg
      181 gcgacgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      241 tgccctggcc caccctggtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      301 ccgaccacat gaagcgccac gacttcttca agagcgccat gcccgagggc tacgtgcagg
      361 agcgcaccat cagcttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg
      421 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      481 acatcctggg ccacaagctg gagtacaact tcaacagcca caacgtgtac atcaccgccg
      541 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacgtg gaggacggca
      601 gcgtgcagct ggccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      661 tgcccgacaa ccactacctg agcacccaga gccgcctgag caaggacccc aacgagaagc
      721 gcgaccacat ggtgctgctg gagttcgtga ccgccgccgg catcacccac ggcatggacg
      781 agctgtacaa gtgaactagt aacttgttta ttgcagctta taatggttac aaataaagca
      841 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
      901 ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt atacgaagtt
      961 atgtcgacgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1021 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1081 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1141 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     1201 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     1261 ttttggaggc ctaggctttt gcaaagaatt cgccaccatg gccaagttga ccagtgccgt
     1321 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     1381 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     1441 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     1501 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     1561 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     1621 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gactgcagcg
     1681 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1741 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1801 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1861 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg aggagggcct
     1921 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     1981 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     2041 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     2101 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     2161 cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc tagcccgcgg
     2221 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     2281 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2341 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2401 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2941 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3181 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3241 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3301 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3361 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3421 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3481 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3541 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3601 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3661 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3721 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3781 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3841 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3901 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3961 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4021 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4081 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4141 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4201 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4261 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4321 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4381 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4441 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4501 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     4561 tatgtatcat acacatacg
//
