LOCUS       sequence-232633-        5035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5035)
  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 5035)
  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-P15
FEATURES             Location/Qualifiers
     source          1..5035
                     /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"
     polyA_signal    2138..2362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2369..2617
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2369..2389
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2540..2559
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2619..2652)
                     /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(2689..2708)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2690..2708)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2795..2812)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2966..3554)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3046..3065)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3725..4516,4517..4585))
                     /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     4348..4367
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4586..4690)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4758..4776
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4814..4836)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4936..4955
                     /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 gactgagtac aagcctactg tgcgtttagc
     1561 caccagggat gatgtgccac gagccgtgag gacactagca gccgcctttg ctgactaccc
     1621 agctacaagg cacaccgtgg accctgacag acacattgaa agagtcacag aacttcagga
     1681 gctgttttta actagggtgg gcctggatat tggcaaagtc tgggtggccg atgatggagc
     1741 agcagttgca gtgtggacca ccccagagtc tgttgaagct ggagctgtct ttgcagaaat
     1801 tggaccaaga atggccgagt tatctggcag caggctggct gctcaacagc agatggaagg
     1861 attattagct ccccacagac ccaaggagcc tgcctggttc ctggcaacag ttggagtttc
     1921 tccagaccat cagggcaagg gcttaggatc tgctgtggtg ctgcctggtg tggaagcagc
     1981 tgagagagct ggggtgccag catttctgga gacatcagct cctagaaacc tgcccttcta
     2041 tgaacggctg ggcttcacag tgactgctga tgtggaggtg cctgagggcc ccagaacctg
     2101 gtgcatgaca agaaaaccag gagcctgact gcagcgactg tgccttctag ttgccagcca
     2161 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2221 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2281 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2341 ggggatgcgg tgggctctat ggctcgagga gggcctattt cccatgattc cttcatattt
     2401 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     2461 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2521 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2581 cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt
     2641 atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag
     2701 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     2761 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2821 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2881 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2941 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3001 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3061 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3121 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3181 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3241 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3301 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3361 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3421 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3481 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3541 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3601 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3661 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3721 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3781 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3841 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3901 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3961 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4021 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4081 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4141 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4201 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4261 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4321 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4381 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4441 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4501 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4561 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4621 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4681 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4741 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     4801 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4861 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4921 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat
     4981 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacg
//
