LOCUS       sequence-228042-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  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 4807)
  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-P05
FEATURES             Location/Qualifiers
     source          1..4807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(64..66,67..780)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(64..66,67..777)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(106..127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(367..386)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     714..735
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    787..908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(824..843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     878..897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(915..948)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        955..1271
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1122..1257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1893..2117
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2124..2372
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2124..2144
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2295..2314
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2374..2407)
                     /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(2444..2463)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2445..2463)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2550..2567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2721..3309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2801..2820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3480..4271,4272..4340))
                     /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     4103..4122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4341..4445)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4513..4531
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4569..4591)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4691..4710
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4791..4807,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4791..4807,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      121 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      241 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      361 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      481 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      541 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      661 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      721 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
      781 actagtaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
      841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
      901 gtatcttagt cgatataact tcgtatagca tacattatac gaagttatgt cgacggtgtg
      961 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     1021 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     1081 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     1141 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     1201 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     1261 gcttttgcaa agaattcgcc accatgactg agtacaagcc tactgtgcgt ttagccacca
     1321 gggatgatgt gccacgagcc gtgaggacac tagcagccgc ctttgctgac tacccagcta
     1381 caaggcacac cgtggaccct gacagacaca ttgaaagagt cacagaactt caggagctgt
     1441 ttttaactag ggtgggcctg gatattggca aagtctgggt ggccgatgat ggagcagcag
     1501 ttgcagtgtg gaccacccca gagtctgttg aagctggagc tgtctttgca gaaattggac
     1561 caagaatggc cgagttatct ggcagcaggc tggctgctca acagcagatg gaaggattat
     1621 tagctcccca cagacccaag gagcctgcct ggttcctggc aacagttgga gtttctccag
     1681 accatcaggg caagggctta ggatctgctg tggtgctgcc tggtgtggaa gcagctgaga
     1741 gagctggggt gccagcattt ctggagacat cagctcctag aaacctgccc ttctatgaac
     1801 ggctgggctt cacagtgact gctgatgtgg aggtgcctga gggccccaga acctggtgca
     1861 tgacaagaaa accaggagcc tgactgcagc gactgtgcct tctagttgcc agccatctgt
     1921 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1981 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2041 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2101 tgcggtgggc tctatggctc gaggagggcc tatttcccat gattccttca tatttgcata
     2161 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     2221 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     2281 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     2341 ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg
     2401 aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta
     2461 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc
     2521 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2581 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2641 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2701 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2761 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2821 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2881 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     2941 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3001 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3061 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3121 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3181 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3241 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3301 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3361 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3421 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3481 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3541 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3601 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3661 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3721 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3781 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3841 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3901 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3961 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4021 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4081 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4141 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4201 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4261 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4321 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4381 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4441 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4501 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     4561 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4621 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     4681 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg
     4741 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
     4801 cactata
//
