LOCUS       sequence-232651-        4564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4564)
  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 4564)
  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-Z02
FEATURES             Location/Qualifiers
     source          1..4564
                     /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..779
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    786..907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(823..842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     877..896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(914..947)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        954..1270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1121..1256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1277..1286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1283..1657
                     /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    1667..1891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1898..2146
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1898..1918
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2069..2088
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2148..2181)
                     /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(2218..2237)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2219..2237)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2324..2341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2495..3083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2575..2594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3254..4045,4046..4114))
                     /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     3877..3896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4115..4219)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4287..4305
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4343..4365)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4465..4484
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc agcgagctga ttaaggagaa catgcacatg aagctgtaca
      121 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
      181 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
      241 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
      301 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
      361 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
      421 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
      481 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
      541 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
      601 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
      661 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
      721 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattgaa
      781 ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
      841 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
      901 tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc gacggtgtgg
      961 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1021 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1081 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     1141 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     1201 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     1261 cttttgcaaa gaattcgcca ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
     1321 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
     1381 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
     1441 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
     1501 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc
     1561 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
     1621 caactgcgtg cacttcgtgg ccgaggagca ggactgactg cagcgactgt gccttctagt
     1681 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1741 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1801 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1861 aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc ccatgattcc
     1921 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     1981 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2041 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2101 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata acttcgtata
     2161 gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg ccggtctccc
     2221 tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac
     2281 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2341 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3241 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3301 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3361 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3421 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3481 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3541 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3601 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3661 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3721 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3781 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3841 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3901 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3961 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4021 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4081 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4141 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4201 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4261 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     4321 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4381 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4441 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     4501 tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca
     4561 tacg
//
