LOCUS       sequence-228056-        4822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mNeptune2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4822)
  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 4822)
  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-P14
FEATURES             Location/Qualifiers
     source          1..4822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..795
                     /codon_start=1
                     /product="brighter version of the far-red fluorescent 
                     protein mNeptune2 with slightly blue-shifted spectra"
                     /label=mNeptune2.5
                     /translation="VSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTNR
                     IKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCDM
                     ALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     polyA_signal    802..923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(839..858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     893..912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(930..963)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        970..1286
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1137..1272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1908..2132
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2139..2387
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2139..2159
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2310..2329
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2389..2422)
                     /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(2459..2478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2460..2478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2565..2582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2736..3324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2816..2835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3495..4286,4287..4355))
                     /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     4118..4137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4356..4460)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4528..4546
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4584..4606)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4706..4725
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4806..4822,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4806..4822,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgatcaag gagaacatgc acaccaagct gtacatggaa
      121 ggcaccgtga acaaccacca cttcaagtgc acccacgaag gggagggcaa gccctacgag
      181 ggcacccaga ccaacaggat taaggtggtg gagggaggcc ccctgccgtt cgcattcgac
      241 atcctggcca cctgctttat gtacgggagc aagaccttca tcaaccacac ccagggcatc
      301 cccgatttct ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
      361 gaagacgggg gcgtgcttac cgttacccag gacaccagcc tccaggacgg ctgcttgatc
      421 tacaacgtca agctcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
      481 acactcggct gggaggccag taccgagacc ctgtaccccg ctgacggcgg cctggaaggc
      541 agatgcgaca tggccctgaa gctcgtgggc gggggccacc tgcactgcaa cctgaagacc
      601 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctttgtggac
      661 cgcagactgg aaagaatcaa ggaggccgac aatgagacct acgtcgagca gcacgaggtg
      721 gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa tggcatggac
      781 gagctgtaca agtgaactag taacttgttt attgcagctt ataatggtta caaataaagc
      841 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
      901 tccaaactca tcaatgtatc ttagtcgata taacttcgta tagcatacat tatacgaagt
      961 tatgtcgacg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     1021 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     1081 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     1141 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     1201 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     1261 tttttggagg cctaggcttt tgcaaagaat tcgccaccat gactgagtac aagcctactg
     1321 tgcgtttagc caccagggat gatgtgccac gagccgtgag gacactagca gccgcctttg
     1381 ctgactaccc agctacaagg cacaccgtgg accctgacag acacattgaa agagtcacag
     1441 aacttcagga gctgttttta actagggtgg gcctggatat tggcaaagtc tgggtggccg
     1501 atgatggagc agcagttgca gtgtggacca ccccagagtc tgttgaagct ggagctgtct
     1561 ttgcagaaat tggaccaaga atggccgagt tatctggcag caggctggct gctcaacagc
     1621 agatggaagg attattagct ccccacagac ccaaggagcc tgcctggttc ctggcaacag
     1681 ttggagtttc tccagaccat cagggcaagg gcttaggatc tgctgtggtg ctgcctggtg
     1741 tggaagcagc tgagagagct ggggtgccag catttctgga gacatcagct cctagaaacc
     1801 tgcccttcta tgaacggctg ggcttcacag tgactgctga tgtggaggtg cctgagggcc
     1861 ccagaacctg gtgcatgaca agaaaaccag gagcctgact gcagcgactg tgccttctag
     1921 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1981 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2041 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2101 caggcatgct ggggatgcgg tgggctctat ggctcgagga gggcctattt cccatgattc
     2161 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     2221 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     2281 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     2341 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat
     2401 agcatacatt atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc
     2461 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     2521 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2581 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2641 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2701 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2761 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2821 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2881 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     2941 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3001 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3061 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3121 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     3181 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3241 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3301 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3361 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3421 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3481 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3541 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3601 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3661 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3721 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3781 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3841 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3901 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3961 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4021 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4081 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4141 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4201 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4261 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4321 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4381 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4441 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4501 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     4561 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4621 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4681 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     4741 atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
     4801 atacgattta ggtgacacta ta
//
