LOCUS       sequence-228017-        3653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mNeptune2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3653)
  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 3653)
  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-14
FEATURES             Location/Qualifiers
     source          1..3653
                     /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..1218
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     970..990
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1141..1160
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1220..1253)
                     /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(1290..1309)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1291..1309)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1396..1413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1567..2155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1647..1666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2326..3117,3118..3186))
                     /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     2949..2968
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3187..3291)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3359..3377
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3415..3437)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3537..3556
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3637..3653,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3637..3653,1)
                     /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 tatgtcgagg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1021 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1081 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1141 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1201 tggaaaggac gaaacaccga taacttcgta tagcatacat tatacgaagt tatggtaccg
     1261 atgcagctag cccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag
     1321 ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1381 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1441 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1501 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1561 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1621 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1681 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1741 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1801 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1861 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1921 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1981 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     2041 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2101 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2161 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2221 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2281 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2341 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2401 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     2461 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     2521 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     2581 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     2641 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     2701 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     2761 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     2821 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     2881 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     2941 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3001 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3061 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3121 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3181 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3241 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     3301 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     3361 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     3421 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3481 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     3541 atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta
     3601 caattaatac ataaccttat gtatcataca catacgattt aggtgacact ata
//
