LOCUS       sequence-228132-        4597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mNeptune2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4597)
  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 4597)
  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-Z14
FEATURES             Location/Qualifiers
     source          1..4597
                     /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"
     regulatory      1293..1302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1299..1673
                     /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    1683..1907
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1914..2162
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1914..1934
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2085..2104
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2164..2197)
                     /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(2234..2253)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2235..2253)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2340..2357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2511..3099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2591..2610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3270..4061,4062..4130))
                     /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     3893..3912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4131..4235)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4303..4321
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4359..4381)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4481..4500
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4581..4597,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4581..4597,1)
                     /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 ggccaagttg accagtgccg
     1321 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     1381 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     1441 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     1501 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     1561 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     1621 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgactgcagc
     1681 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1741 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1801 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1861 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gaggagggcc
     1921 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1981 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     2041 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     2101 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     2161 ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg
     2221 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat
     2281 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2341 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2401 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2461 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2521 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2581 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2641 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2701 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2761 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2821 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2881 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2941 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3001 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3061 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3121 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3181 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3241 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3301 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3361 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3421 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3481 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3541 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3601 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3661 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3721 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3781 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3841 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3901 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3961 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4021 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4081 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4141 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4201 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4261 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4321 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4381 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4441 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     4501 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
     4561 ttatgtatca tacacatacg atttaggtga cactata
//
