LOCUS       sequence-373423-        3488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry clone with ORF encoding plasma membrane-targeted EGFP-GPI 
            fusion protein flanked by Gateway recombination sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3488)
  AUTHORS   Roure A, Rothbacher U, Robin F, Kalmar E, Ferone G, Lamy C, Missero 
            C, Mueller F, Lemaire P
  TITLE     A multicassette Gateway vector set for high throughput and 
            comparative analyses in ciona and vertebrate embryos.
  JOURNAL   PLoS One. 2007. 2(9):e916.
  PUBMED    17878951
REFERENCE   2  (bases 1 to 3488)
  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: "PLoS One. 
            2007. 2(9):e916."
COMMENT     Sequence Label: pENT-L1-Kozak-EGFP-GPI-stop-L2
FEATURES             Location/Qualifiers
     source          1..3488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     104..121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(280..307)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(399..485)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     534..556
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     548..565
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     549..565
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    581..680
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     sig_peptide     689..760
                     /codon_start=1
                     /product="signal sequence of human preproinsulin"
                     /label=insulin signal sequence
                     /translation="MALWMRLLPLLALLALWGPDPAAA"
     regulatory      776..785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(782..784,785..787,788..1495)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(782..784,785..787,788..1495)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(827..848)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1088..1107)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1435..1456
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1625..1724)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1741..1760)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1742..1760)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1765..1781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1765..1781)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1883..1902)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1894..2703
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1961..1980)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2850..3438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3339..3358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
       61 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      121 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      181 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      241 tacgcgtacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg
      301 gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
      361 ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg
      421 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt
      481 atttgatgcc tggcagttcc ctactctcgc gttaacgcta gcatggatgt tttcccagtc
      541 acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc caaataatga ttttattttg
      601 actgatagtg acctgttcgt tgcaacaaat tgatgagcaa tgctttttta taatgccaac
      661 tttgtacaaa aaagcaggct cagaaaaaat ggccctgtgg atgcgcctcc tgcccctgct
      721 ggcgctgctg gccctctggg gacctgaccc agccgcagcc ggggatccac cggtcgccac
      781 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      841 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      901 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      961 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1021 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1081 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1141 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1201 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1261 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1321 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1381 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1441 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacagtgt
     1501 cgacacgcgt ccaaataaag gaagtggaac cacttcaggt actacccgtc ttctatctgg
     1561 gcacacgtgt ttcacgttga caggtttgct tgggacgcta gtaaccatgg gcttgctgac
     1621 ttagacccag ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt
     1681 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc
     1741 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     1801 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg
     1861 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     1921 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     1981 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     2041 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     2101 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     2161 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     2221 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     2281 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     2341 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     2401 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     2461 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     2521 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     2581 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     2641 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     2701 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
     2761 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     2821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3481 tggccttt
//
