LOCUS       sequence-345191-        4438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for amplification of EGFP C-terminal tagging cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4438)
  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 4438)
  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-CT-Tagging
FEATURES             Location/Qualifiers
     source          1..4438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     152..171
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     411..432
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(483..516)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(542..589)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     585..603
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             1512..1520
                     /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)"
     protein_bind    complement(1564..1597)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(1653..2108)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1790..1811)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2002..2021
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(2222..3037)
                     /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             complement(2222..3037)
                     /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     2945..2964
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3038..3140)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3255..3843
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3744..3763
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3967..4191
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(4196..4438,1..471,472..474,475..477))
                     /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             complement(join(4199..4438,1..471,472..474,475..477))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4241..4262)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
       61 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
      121 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
      181 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
      241 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
      301 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
      361 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
      421 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatcga
      481 ggataacttc gtatagcata cattatacga agttatatcc ggatatagtt cctcctttca
      541 gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag
      601 cggtggcagc agccaactca gcttcctttc gggctttgaa gcttaccaat gcttaataag
      661 agaagccccg atttctgcga tctgacggtt acgctcgtcc atcgtcgcct gggagcctgt
      721 cgtatagatt accacaatac gtgagggttt cccatccgga ccaagtgctg caataatgcc
      781 acgagaaccg cgttctcccg ccccagactt atccgcaata aaccatccgg ctggcagggc
      841 agagcgcagc agcggcccgg ccactttatc tgcctccatc caatcgataa gttgctggcg
      901 agatgccagg gttaaaagct ctccagtcag caatttacgc agcgttgtgg ccatagcgac
      961 gggcatcgtc gtgtcacgtt cgtcattggg gatagcttcg tttaattccg gctcccagcg
     1021 gtctaaacga gtgacgtgat cgcccatgtt gtgaaggaat gccgtcagct ccttcggacc
     1081 gccaatggtc gtaagcagaa ggttggcggc ggtgttatca gacatcgtaa tagcggcact
     1141 acacagttca cgaactgtca tgccatcagt caaatgcttc tctgttacgg gggagtattc
     1201 cacaagatca ttctgggagt agtggatacg acggcccagt tgctcttgac ctgcatcgat
     1261 ggagcttaaa accgcgccgc acaacaacac tttgaaggta ctcatcatgg ggaaacgttc
     1321 ttcagggcgg aagctctcta agatttttcc ggaattaagg tccaactcaa tgtaaccgac
     1381 acgagcacca agttgatcct ctgcatcctt aactttgacc agcgtttctg gatgggcgaa
     1441 tactggcaaa cagaatgcag caaaaaaggg gatcagcgcc acacggaaat gttgaatact
     1501 cattatgtct aacctcctta cgtatcgcat attatatgtg aatcacagtg atatgtaagt
     1561 attataactt cgtatagcat acattatacg aagttatggc actgccgccg ctacctccgc
     1621 ttccaccgga acctcctgag acgaattcgt taaaattgta agcgttaata ttttgttaaa
     1681 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     1741 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     1801 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     1861 gggcgctggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     1921 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     1981 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2041 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2101 gggcgcgtaa catcatgttt gacagcttat catcgaattt ctgccattca tccgcttatt
     2161 atcacttatt caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa
     2221 atcagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2281 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2341 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2401 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2461 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     2521 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2581 gcgcctgagc gagtcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2641 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2701 attcttctaa tacctggaat gctgttttcc ctgggatcgc agtggtgagt aaccatgcat
     2761 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2821 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2881 acaactctgg cgcatcgggc ttcccataca atcggtagat tgtcgcacct gattgcccga
     2941 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3001 gccttgagca agacgtttcc cgttgaatat ggctcatttt agcttcctta gctcctgaaa
     3061 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     3121 aacctcttac gtgccgatca tcacaaaagg atctaggtga agatcctttt tgataatctc
     3181 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3241 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3301 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3361 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3421 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3481 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3541 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3601 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3661 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3721 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3781 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3841 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3901 atgttctttc ctgcgttatc ccctgattct gtggataacc gtgtcagaat tcgtctcatg
     3961 acagcgccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
     4021 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
     4081 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
     4141 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gaagcttact
     4201 tgtatagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     4261 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     4321 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     4381 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgtt
//
