LOCUS       sequence-232632-        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"
     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..812
                     /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    819..940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(856..875)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     910..929
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(947..980)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        987..1303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1154..1289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1925..2149
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2156..2404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2156..2176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2327..2346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2406..2439)
                     /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(2476..2495)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2477..2495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2582..2599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2753..3341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2833..2852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3512..4303,4304..4372))
                     /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     4135..4154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4373..4477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4545..4563
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4601..4623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4723..4742
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gatcaaggag aacatgcaca
      121 cgaagctgta catggaaggc accgtgaaca accaccactt caagtgcacc cacgaagggg
      181 agggcaagcc ctacgagggc acccagacca acaggattaa ggtggtggag ggaggccccc
      241 tgccgttcgc attcgacatc ctggccacct gctttatgta cgggagcaag accttcatca
      301 accacaccca gggcatcccc gatttcttta agcagtcctt ccctgagggc ttcacatggg
      361 agagagtcac cacatacgaa gacgggggcg tgcttaccgt tacccaggac accagcctcc
      421 aggacggctg cttgatctac aacgtcaagc tcagaggggt gaacttccca tccaacggcc
      481 ctgtgatgca gaagaaaaca ctcggctggg aggccagtac cgagaccctg taccccgctg
      541 acggcggcct ggaaggcaga tgcgacatgg ccctgaagct cgtgggcggg ggccacctgc
      601 actgcaacct gaagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg
      661 gcgtctactt tgtggaccgc agactggaaa gaatcaagga ggccgacaat gagacctacg
      721 tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca
      781 aacttaatgg catggacgag ctgtacaagt gaactagtaa cttgtttatt gcagcttata
      841 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
      901 attctagttg tggtttgtcc aaactcatca atgtatctta gtcgatataa cttcgtatag
      961 catacattat acgaagttat gtcgacggtg tggaaagtcc ccaggctccc cagcaggcag
     1021 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     1081 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     1141 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     1201 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     1261 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagaattcg ccaccatgac
     1321 tgagtacaag cctactgtgc gtttagccac cagggatgat gtgccacgag ccgtgaggac
     1381 actagcagcc gcctttgctg actacccagc tacaaggcac accgtggacc ctgacagaca
     1441 cattgaaaga gtcacagaac ttcaggagct gtttttaact agggtgggcc tggatattgg
     1501 caaagtctgg gtggccgatg atggagcagc agttgcagtg tggaccaccc cagagtctgt
     1561 tgaagctgga gctgtctttg cagaaattgg accaagaatg gccgagttat ctggcagcag
     1621 gctggctgct caacagcaga tggaaggatt attagctccc cacagaccca aggagcctgc
     1681 ctggttcctg gcaacagttg gagtttctcc agaccatcag ggcaagggct taggatctgc
     1741 tgtggtgctg cctggtgtgg aagcagctga gagagctggg gtgccagcat ttctggagac
     1801 atcagctcct agaaacctgc ccttctatga acggctgggc ttcacagtga ctgctgatgt
     1861 ggaggtgcct gagggcccca gaacctggtg catgacaaga aaaccaggag cctgactgca
     1921 gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1981 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2041 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2101 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc tcgaggaggg
     2161 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2221 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2281 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2341 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2401 caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg
     2461 cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc
     2521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3181 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4501 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4561 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4621 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4681 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     4741 tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa
     4801 ccttatgtat catacacata cg
//
