LOCUS       sequence-243810-        3629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3629)
  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 3629)
  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-07
FEATURES             Location/Qualifiers
     source          1..3629
                     /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..788
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      333..342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    795..916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(832..851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     886..905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(923..956)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        963..1211
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     963..983
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1134..1153
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1213..1246)
                     /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(1283..1302)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1284..1302)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1389..1406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1560..2148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1640..1659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2319..3110,3111..3179))
                     /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     2942..2961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3180..3284)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3352..3370
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3408..3430)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3530..3549
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagga taacatggcc tctctcccag
      121 cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac atggtgggtc
      181 agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc accaagggtg
      241 acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc catcagtacc
      301 tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc tccggatacc
      361 aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac taccgctaca
      421 cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt ttccctgctg
      481 acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt
      541 accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact ggaaatggca
      601 agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg gcggctaact
      661 atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac tccaagaccg
      721 agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg gacgagctgt
      781 acaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      841 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      901 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg
      961 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     1021 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     1081 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     1141 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     1201 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     1261 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     1321 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1381 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1441 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1501 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1561 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1621 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1681 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1741 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1801 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1861 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1921 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1981 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2041 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2101 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2161 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2221 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2281 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2341 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2401 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2461 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2521 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2581 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2641 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2701 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2761 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2821 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     2881 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     2941 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3001 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3061 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3121 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3181 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3241 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3301 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3361 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3421 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3481 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     3541 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     3601 tacataacct tatgtatcat acacatacg
//
