LOCUS       sequence-232654-        4582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4582)
  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 4582)
  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-Z05
FEATURES             Location/Qualifiers
     source          1..4582
                     /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             join(81..83,84..797)
                     /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(81..83,84..794)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(123..144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(384..403)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     731..752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    804..925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(841..860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     895..914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(932..965)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        972..1288
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1139..1274
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1295..1304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1301..1675
                     /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    1685..1909
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1916..2164
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1916..1936
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2087..2106
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2166..2199)
                     /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(2236..2255)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2237..2255)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2342..2359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2513..3101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2593..2612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3272..4063,4064..4132))
                     /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     3895..3914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4133..4237)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4305..4323
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4361..4383)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4483..4502
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      181 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      241 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
      301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
      541 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      661 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
      721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      781 acgagctgta caagtaaact agtaacttgt ttattgcagc ttataatggt tacaaataaa
      841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
      901 tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac attatacgaa
      961 gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     1021 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     1081 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     1141 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     1201 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     1261 cttttttgga ggcctaggct tttgcaaaga attcgccacc atggccaagt tgaccagtgc
     1321 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     1381 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     1441 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
     1501 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
     1561 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
     1621 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgactgca
     1681 gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1741 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1801 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1861 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc tcgaggaggg
     1921 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     1981 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2041 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2101 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2161 caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg
     2221 cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc
     2281 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2341 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2401 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2461 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2521 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2581 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2641 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2701 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2761 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2821 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2881 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2941 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3001 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3061 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3121 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3181 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3241 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3301 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3361 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3421 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3481 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3541 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3601 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3661 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3721 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3781 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3841 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3901 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3961 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4021 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4081 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4141 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4201 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4261 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4321 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4381 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4441 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     4501 tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa
     4561 ccttatgtat catacacata cg
//
