LOCUS       sequence-133614-        4695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  template for C-terminal eGFP tagging, Puromycin-resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4695)
  AUTHORS   Kunzelmann S, Bottcher R, Schmidts I, Forstemann K
  TITLE     A Comprehensive Toolbox for Genome Editing in Cultured Drosophila 
            melanogaster Cells.
  JOURNAL   G3 (Bethesda). 2016 Jun 1;6(6):1777-85. doi: 10.1534/g3.116.028241.
  PUBMED    27172193
REFERENCE   2  (bases 1 to 4695)
  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"; doi: "10.1534/g3.116"; 
            journalName: "G3 (Bethesda)"; date: "2016-06-1- 1"; volume: "6"; 
            issue: "6"; pages: "1777-85"
COMMENT     Sequence Label: pSK23
FEATURES             Location/Qualifiers
     source          1..4695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(358..360,361..363,364..1077)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(358..360,361..363,364..1074)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(403..424)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(664..683)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1011..1032
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1084..1117)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(2059..2092)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(join(2527..2533,2534..2551,2552..2557))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2572..2593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2713..2730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2884..3472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2964..2983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3643..4434,4435..4503))
                     /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     4266..4285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4504..4608)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4676..4694
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagatg
      361 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      421 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      481 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      541 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      601 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      661 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      721 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      781 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      841 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      901 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      961 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1021 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaact
     1081 agtgaagttc ctatactttc tagagaatag gaacttcaaa taaacatatg ggcgcgccct
     1141 gttggaatat actattcaac ctacaaaaat aacgttaaac aacactactt tatatttgat
     1201 atgaatggcc acacctttta tgccataaaa catattgtaa gagaatacca ctctttttat
     1261 tccttctttc cttcttgtac gttttttgct gtgagtaggt cgtggtgctg gtgttgcagt
     1321 tgaaataact taaaatataa atcataaaac tcaaacataa acttgactat ttatttattt
     1381 attaagaaag gaggtaccgg tggcgcgcca tgaccgagta caagcctaca gtgcgactgg
     1441 ccacacgcga tgacgttccc agagccgttc ggacgttggc tgccgcgttt gcggactatc
     1501 ccgcaactcg ccatactgtg gaccctgatc gccacatcga gagggtgact gagcttcagg
     1561 agctgttcct gaccagggta gggctggaca taggcaaggt ctgggtggcc gacgatggag
     1621 ccgctgttgc ggtgtggact accccagaga gcgtggaagc aggggctgtc tttgccgaaa
     1681 ttgggccaag gatggccgaa ctgtctgggt cccgtttggc tgcccaacag cagatggaag
     1741 gactgcttgc cccacatcga cccaaggagc ctgcatggtt cctcgctacc gtgggtgtga
     1801 gcccagatca ccagggcaaa ggtctgggat cagccgtcgt tctgcctggc gtcgaggctg
     1861 ctgagagagc aggagtaccg gcattcctcg aaaccagtgc tccacggaac ctcccgttct
     1921 acgagagact gggctttacg gtcacagccg atgtggaagt gcccgaaggc cctagaacct
     1981 ggtgcatgac acggaaaccc ggtgcatgaa aattaaatat aaataaagct attaaaataa
     2041 attactatct aacatatgga agttcctata ctttctagag aataggaact tcatctttct
     2101 agaagatctc ctacaatatt ctcagctgcc atggaaaatc gatgttcttc ttttattctc
     2161 tcaagatttt caggctgtat attaaaactt atattaagaa ctatgctaac cacctcatca
     2221 ggaaccgttg taggtggcgt gggttttctt ggcaatcgac tctcatgaaa actacgagct
     2281 aaatattcaa tatgttcctc ttgaccaact ttattctgca ttttttttga acgaggttta
     2341 gagcaagctt caggaaactg agacaggaat tttattaaaa atttaaattt tgaagaaagt
     2401 tcagggttaa tagcatccat tttttgcttt gcaagttcct cagcattctt aacaaaagac
     2461 gtctcttttg acatgtttaa agtttaaacc tcctgtgtga aattattatc cgctcataat
     2521 tccacacatt atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2581 ctaactcaca ttaattgcgt tgcgctcact gccaattgct ttccagtcgg gaaacctgtc
     2641 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2701 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2761 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2821 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2881 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2941 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3001 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3061 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3121 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3181 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3241 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3301 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3361 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3421 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3481 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3541 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3601 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3661 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3721 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3781 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3841 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3901 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3961 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4021 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4081 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4141 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4201 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4261 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4321 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4381 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4441 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4501 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4561 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4621 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4681 gcgtatcacg aggcc
//
