LOCUS       sequence-232666-        4597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mNeptune2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4597)
  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 4597)
  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-Z14
FEATURES             Location/Qualifiers
     source          1..4597
                     /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..1303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1154..1289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1316..1690
                     /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    1700..1924
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1931..2179
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1931..1951
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2102..2121
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2181..2214)
                     /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(2251..2270)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2252..2270)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2357..2374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2528..3116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2608..2627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3287..4078,4079..4147))
                     /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     3910..3929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4148..4252)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4320..4338
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4376..4398)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4498..4517
                     /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 gtcgacggtg tggaaagtcc ccaggctccc cagcaggcag
     1021 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     1081 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     1141 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     1201 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     1261 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagaattcg ccaccatggc
     1321 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     1381 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     1441 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     1501 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     1561 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     1621 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     1681 gcaggactga ctgcagcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1741 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1801 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1861 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1921 atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     1981 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     2041 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     2101 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     2161 gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag ttatggtacc
     2221 gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     2281 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3661 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4081 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4261 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     4321 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     4381 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4441 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     4501 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     4561 acaattaata cataacctta tgtatcatac acatacg
//
