LOCUS       sequence-232514-        3866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3866)
  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 3866)
  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-15
FEATURES             Location/Qualifiers
     source          1..3866
                     /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..1448
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1200..1220
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1371..1390
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1520..1539)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1521..1539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1626..1643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1797..2385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1877..1896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2556..3347,3348..3416))
                     /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     3179..3198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3417..3521)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3589..3607
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3645..3667)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3767..3786
                     /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 tatgtcgagg
     1201 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     1261 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     1321 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     1381 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     1441 gaaacaccga taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag
     1501 cccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     1561 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1621 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1681 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1741 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1801 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1861 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1921 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1981 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2041 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2101 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2161 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2221 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2281 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2341 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2401 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2461 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2521 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2581 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2641 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2701 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2761 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2821 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2881 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2941 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3001 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3061 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3121 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3181 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3241 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3301 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3361 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3421 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3481 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3541 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3601 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3661 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     3721 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     3781 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     3841 ataaccttat gtatcataca catacg
//
