LOCUS       sequence-232667-        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"
     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..1025
                     /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    1032..1153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1069..1088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1123..1142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1160..1193)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1200..1516
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1367..1502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1523..1532
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1529..1903
                     /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    1913..2137
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2144..2392
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2144..2164
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2315..2334
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2394..2427)
                     /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(2464..2483)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2465..2483)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2570..2587)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2741..3329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2821..2840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3500..4291,4292..4360))
                     /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     4123..4142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4361..4465)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4533..4551
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4589..4611)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4711..4730
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatct gtagcaggtc atgcctctgg cagccccgca ttcgggaccg
      121 cctctcattc gaattgcgaa catgaagaga tccacctcgc cggctcgatc cagccgcatg
      181 gcgcgcttct ggtcgtcagc gaacatgatc atcgcgtcat ccaggccagc gccaacgccg
      241 cggaatttct gaatctcgga agcgtactcg gcgttccgct cgccgagatc gacggcgatc
      301 tgttgatcaa gatcctgccg catctcgatc ccaccgccga aggcatgccg gtcgcggtgc
      361 gctgccggat cggcaatccc tctacggagt actgcggtct gatgcatcgg cctccggaag
      421 gcgggctgat catcgaactc gaacgtgccg gcccgtcgat cgatctgtca ggcacgctgg
      481 cgccggcgct ggagcggatc cgcacggcgg gttcactgcg cgcgctgtgc gatgacaccg
      541 tgctgctgtt tcagcagtgc accggctacg accgggtgat ggtgtatcgt ttcgatgagc
      601 aaggccacgg cctggtattc tccgagtgcc atgtgcctgg gctcgaatcc tatttcggca
      661 accgctatcc gtcgtcgact gtcccgcaga tggcgcggca gctgtacgtg cggcagcgcg
      721 tccgcgtgct ggtcgacgtc acctatcagc cggtgccgct ggagccgcgg ctgtcgccgc
      781 tgaccgggcg cgatctcgac atgtcgggct gcttcctgcg ctcgatgtcg ccgtgccatc
      841 tgcagttcct gaaggacatg ggcgtgcgcg ccaccctggc ggtgtcgctg gtggtcggcg
      901 gcaagctgtg gggcctggtt gtctgtcacc attatctgcc gcgcttcatc cgtttcgagc
      961 tgcgggcgat ctgcaaacgg ctcgccgaaa ggatcgcgac gcggatcacc gcgcttgaga
     1021 gctgaactag taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     1141 tcaatgtatc ttagtcgata taacttcgta tagcatacat tatacgaagt tatgtcgacg
     1201 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     1261 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     1321 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     1381 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     1441 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     1501 cctaggcttt tgcaaagaat tcgccaccat ggccaagttg accagtgccg ttccggtgct
     1561 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     1621 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     1681 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     1741 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     1801 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     1861 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgactgcagc gactgtgcct
     1921 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     1981 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2041 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2101 aatagcaggc atgctgggga tgcggtgggc tctatggctc gaggagggcc tatttcccat
     2161 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     2221 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2281 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2341 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgataactt
     2401 cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg gatctgccgg
     2461 tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg
     2521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3181 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3781 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4441 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4501 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4561 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     4621 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     4681 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4741 accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca
     4801 tacacatacg
//
