LOCUS       sequence-228133-        4810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4810)
  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 4810)
  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-Z15
FEATURES             Location/Qualifiers
     source          1..4810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..1008
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="VAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDHR
                     VIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEY
                     CGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGY
                     DRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVTY
                     QPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVV
                     CHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    1015..1136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1052..1071)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1106..1125
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1143..1176)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1183..1499
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1350..1485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1506..1515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1512..1886
                     /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    1896..2120
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2127..2375
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2127..2147
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2298..2317
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2377..2410)
                     /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(2447..2466)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2448..2466)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2553..2570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2724..3312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2804..2823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3483..4274,4275..4343))
                     /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     4106..4125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4344..4448)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4516..4534
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4572..4594)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4694..4713
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4794..4810,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4794..4810,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tctgtagcag gtcatgcctc tggcagcccc gcattcggga ccgcctctca ttcgaattgc
      121 gaacatgaag agatccacct cgccggctcg atccagccgc atggcgcgct tctggtcgtc
      181 agcgaacatg atcatcgcgt catccaggcc agcgccaacg ccgcggaatt tctgaatctc
      241 ggaagcgtac tcggcgttcc gctcgccgag atcgacggcg atctgttgat caagatcctg
      301 ccgcatctcg atcccaccgc cgaaggcatg ccggtcgcgg tgcgctgccg gatcggcaat
      361 ccctctacgg agtactgcgg tctgatgcat cggcctccgg aaggcgggct gatcatcgaa
      421 ctcgaacgtg ccggcccgtc gatcgatctg tcaggcacgc tggcgccggc gctggagcgg
      481 atccgcacgg cgggttcact gcgcgcgctg tgcgatgaca ccgtgctgct gtttcagcag
      541 tgcaccggct acgaccgggt gatggtgtat cgtttcgatg agcaaggcca cggcctggta
      601 ttctccgagt gccatgtgcc tgggctcgaa tcctatttcg gcaaccgcta tccgtcgtcg
      661 actgtcccgc agatggcgcg gcagctgtac gtgcggcagc gcgtccgcgt gctggtcgac
      721 gtcacctatc agccggtgcc gctggagccg cggctgtcgc cgctgaccgg gcgcgatctc
      781 gacatgtcgg gctgcttcct gcgctcgatg tcgccgtgcc atctgcagtt cctgaaggac
      841 atgggcgtgc gcgccaccct ggcggtgtcg ctggtggtcg gcggcaagct gtggggcctg
      901 gttgtctgtc accattatct gccgcgcttc atccgtttcg agctgcgggc gatctgcaaa
      961 cggctcgccg aaaggatcgc gacgcggatc accgcgcttg agagctgaac tagtaacttg
     1021 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1081 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg
     1141 atataacttc gtatagcata cattatacga agttatgtcg acggtgtgga aagtccccag
     1201 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     1261 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     1321 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     1381 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     1441 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     1501 aattcgccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     1561 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     1621 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     1681 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     1741 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     1801 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     1861 acttcgtggc cgaggagcag gactgactgc agcgactgtg ccttctagtt gccagccatc
     1921 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1981 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2041 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2101 ggatgcggtg ggctctatgg ctcgaggagg gcctatttcc catgattcct tcatatttgc
     2161 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     2221 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2281 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2341 tggctttata tatcttgtgg aaaggacgaa acaccgataa cttcgtatag catacattat
     2401 acgaagttat ggtaccgatg cagctagccc gcggatctgc cggtctccct atagtgagtc
     2461 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     2521 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2581 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2641 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2701 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2761 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2821 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2881 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     2941 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3001 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3061 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3121 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3181 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3241 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3301 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3361 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3421 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3481 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3541 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3601 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3661 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     3721 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     3781 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3841 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3901 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3961 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4021 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4081 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4141 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4201 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4261 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4321 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4381 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4441 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     4501 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     4561 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     4621 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4681 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     4741 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg
     4801 tgacactata
//
