LOCUS       sequence-232513-        3653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mNeptune2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3653)
  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 3653)
  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-14
FEATURES             Location/Qualifiers
     source          1..3653
                     /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..812
                     /codon_start=1
                     /product="brighter version of the far-red fluorescent 
                     protein mNeptune2 with slightly blue-shifted spectra"
                     /label=mNeptune2.5
                     /translation="VSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTNR
                     IKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCDM
                     ALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     polyA_signal    819..940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(856..875)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     910..929
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(947..980)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        987..1235
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     987..1007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1158..1177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1237..1270)
                     /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(1307..1326)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1308..1326)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1413..1430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1584..2172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1664..1683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2343..3134,3135..3203))
                     /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     2966..2985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3204..3308)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3376..3394
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3432..3454)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3554..3573
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gatcaaggag aacatgcaca
      121 cgaagctgta catggaaggc accgtgaaca accaccactt caagtgcacc cacgaagggg
      181 agggcaagcc ctacgagggc acccagacca acaggattaa ggtggtggag ggaggccccc
      241 tgccgttcgc attcgacatc ctggccacct gctttatgta cgggagcaag accttcatca
      301 accacaccca gggcatcccc gatttcttta agcagtcctt ccctgagggc ttcacatggg
      361 agagagtcac cacatacgaa gacgggggcg tgcttaccgt tacccaggac accagcctcc
      421 aggacggctg cttgatctac aacgtcaagc tcagaggggt gaacttccca tccaacggcc
      481 ctgtgatgca gaagaaaaca ctcggctggg aggccagtac cgagaccctg taccccgctg
      541 acggcggcct ggaaggcaga tgcgacatgg ccctgaagct cgtgggcggg ggccacctgc
      601 actgcaacct gaagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg
      661 gcgtctactt tgtggaccgc agactggaaa gaatcaagga ggccgacaat gagacctacg
      721 tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca
      781 aacttaatgg catggacgag ctgtacaagt gaactagtaa cttgtttatt gcagcttata
      841 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
      901 attctagttg tggtttgtcc aaactcatca atgtatctta gtcgatataa cttcgtatag
      961 catacattat acgaagttat gtcgaggagg gcctatttcc catgattcct tcatatttgc
     1021 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     1081 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     1141 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     1201 tggctttata tatcttgtgg aaaggacgaa acaccgataa cttcgtatag catacattat
     1261 acgaagttat ggtaccgatg cagctagccc gcggatctgc cggtctccct atagtgagtc
     1321 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     1381 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1441 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1501 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1561 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1621 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1681 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1741 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1801 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1861 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1921 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1981 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2041 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2101 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2161 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2221 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2281 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2341 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2401 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2461 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2521 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2581 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2641 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2701 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2761 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2821 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2881 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2941 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3001 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3061 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3121 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3181 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3301 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3361 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     3421 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3481 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3541 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     3601 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acg
//
