LOCUS       sequence-227995-        3638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3638)
  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 3638)
  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-05
FEATURES             Location/Qualifiers
     source          1..3638
                     /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..1203
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     955..975
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1126..1145
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1205..1238)
                     /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(1275..1294)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1276..1294)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1381..1398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1552..2140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1632..1651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2311..3102,3103..3171))
                     /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     2934..2953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3172..3276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3344..3362
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3400..3422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3522..3541
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3622..3638,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3622..3638,1)
                     /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 cgaggagggc
      961 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     1021 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     1081 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     1141 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     1201 accgataact tcgtatagca tacattatac gaagttatgg taccgatgca gctagcccgc
     1261 ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca
     1321 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1381 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1441 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1501 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1561 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1621 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1681 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     1741 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1801 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     1861 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1921 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1981 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2041 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2101 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2161 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2221 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2281 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2401 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2461 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2521 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2821 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3061 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3121 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3181 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3241 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3301 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     3361 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3421 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3481 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     3541 actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac
     3601 cttatgtatc atacacatac gatttaggtg acactata
//
