LOCUS       sequence-335648-        4429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for amplification of EGFP N-terminal tagging cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4429)
  AUTHORS   Matinyan N, Karkhanis MS, Gonzalez Y, Jain A, Saltzman A, 
            Malovannaya A, Sarrion-Perdigones A, Dierick HA, Venken KJT
  TITLE     Multiplexed drug-based selection and counterselection genetic 
            manipulations in Drosophila.
  JOURNAL   Cell Rep. 2021 Sep 14;36(11):109700. doi: 
            10.1016/j.celrep.2021.109700.
  PUBMED    34525356
REFERENCE   2  (bases 1 to 4429)
  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: "Cell Rep.";
            date: "2021-09-14"; volume: "36(11):109700. doi"; pages: " 
            10.1016/j.celrep.2021.109700"
COMMENT     Sequence Label: pEGFP-NT-Tagging
FEATURES             Location/Qualifiers
     source          1..4429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(144..732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(224..243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        847..949
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             950..1765
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             950..1765
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1023..1042)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1879..2334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1966..1985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2176..2197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2358..2569
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(2571..2573,2574..2576,2577..3287)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2571..2573,2574..2576,2577..3287)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2616..2637)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2877..2896)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3224..3245
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3294..3327
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     RBS             3372..3380
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(4289..4307)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4303..4350
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     protein_bind    4376..4409
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 agcggaggta gcggcggcag cgctgtcatg agacgaattc tgacacggtt atccacagaa
       61 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      121 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      181 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      241 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      301 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      361 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      421 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      481 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      541 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      601 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      661 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
      721 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
      781 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      841 ttgtgatgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
      901 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tgagccatat
      961 tcaacgggaa acgtcttgct caaggccgcg attaaattcc aacatggatg ctgatttata
     1021 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct accgattgta
     1081 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     1141 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     1201 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tcccagggaa
     1261 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1321 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1381 tcgcgtattt cgactcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1441 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1501 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     1561 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     1621 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     1681 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     1741 tttgatgctc gatgagtttt tctgattttt tttaaggcag ttattggtgc ccttaaacgc
     1801 ctggttgcta cgcctgaata agtgataata agcggatgaa tggcagaaat tcgatgataa
     1861 gctgtcaaac atgatgttac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1921 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1981 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2041 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2101 atggttcacg tagtgggcca gcgccctgat agacggtttt tcgccctttg acgttggagt
     2161 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2221 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2281 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca attttaacga
     2341 attcgtctca ggagtatggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2401 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2461 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2521 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacaga atggtgagca
     2581 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2641 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2701 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2761 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2821 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2881 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2941 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3001 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3061 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3121 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3181 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3241 tcgtgaccgc cgccgggatc actctcggca tggatgagct gtacaagagt gccataactt
     3301 cgtataatgt atgctatacg aagttataat acttgacata tcactgtgat tcacatataa
     3361 tatgcgatac gtaaggaggt cagacataat gagtattcaa catttccgtg tggcgctgat
     3421 cccctttttt gctgcattct gtttgccagt attcgcccat ccagaaacgc tggtcaaagt
     3481 taaggatgca gaggatcaac ttggtgctcg tgtcggttac attgagttgg accttaattc
     3541 cggaaaaatc ttagagagct tccgccctga agaacgtttc cccatgatga gtaccttcaa
     3601 agtgttgttg tgcggcgcgg ttttaagctc catcgatgca ggtcaagagc aactgggccg
     3661 tcgtatccac tactcccaga atgatcttgt ggaatactcc cccgtaacag agaagcattt
     3721 gactgatggc atgacagttc gtgaactgtg tagtgccgct attacgatgt ctgataacac
     3781 cgccgccaac cttctgctta cgaccattgg cggtccgaag gagctgacgg cattccttca
     3841 caacatgggc gatcacgtca ctcgtttaga ccgctgggag ccggaattaa acgaagctat
     3901 ccccaatgac gaacgtgaca cgacgatgcc cgtcgctatg gccacaacgc tgcgtaaatt
     3961 gctgactgga gagcttttaa ccctggcatc tcgccagcaa cttatcgatt ggatggaggc
     4021 agataaagtg gccgggccgc tgctgcgctc tgccctgcca gccggatggt ttattgcgga
     4081 taagtctggg gcgggagaac gcggttctcg tggcattatt gcagcacttg gtccggatgg
     4141 gaaaccctca cgtattgtgg taatctatac gacaggctcc caggcgacga tggacgagcg
     4201 taaccgtcag atcgcagaaa tcggggcttc tcttattaag cattggtaag cttcaaagcc
     4261 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     4321 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatataac
     4381 ttcgtataat gtatgctata cgaagttatc cctcggaggt tccggtgga
//
