LOCUS       sequence-492418-        6149 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  mammalian expression construct for human EGF-like repeats and 
            discoidin I-like domains 3 C-terminally tagged with EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6149)
  TITLE     GFP-tagged lysosomal proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6149)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: hEDIL3-EGFP
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2089..2098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2095..2097,2098..2100,2101..2814)
                     /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(2095..2097,2098..2100,2101..2811)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2140..2161)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2401..2420)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2748..2769
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2937..3058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2974..2993)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3028..3047
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3065..3520)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3202..3223)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3414..3433
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3547..3651
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3653..4010
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3653..3844
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3676..3696)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3861..3996
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4045..4839
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4045..4839
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4099..4118)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4709..4728
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5027..5046)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5071..5118
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5447..6035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5936..5955
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttgcc accatgaagc gctcggtagc cgtctggctc
      661 ttggtcgggc tcagcctcgg tgtcccccag ttcggcaaag gtgatatttg tgatcccaat
      721 ccatgtgaaa atggaggtat ctgtttgcca ggattggctg atggttcctt ttcctgtgag
      781 tgtccagatg gcttcacaga ccccaactgt tctagtgttg tggaggttgc atcagatgaa
      841 gaagaaccaa cttcagcagg tccctgcact cctaatccat gccataatgg aggaacctgt
      901 gaaataagtg aagcataccg aggggataca ttcataggct atgtttgtaa atgtccccga
      961 ggatttaatg ggattcactg tcagcacaac ataaatgaat gcgaagttga gccttgcaaa
     1021 aatggtggaa tatgtacaga tcttgttgct aactattcct gtgagtgccc aggcgaattt
     1081 atgggaagaa attgtcaata caaatgctca ggcccactgg gaattgaagg tggaattata
     1141 tcaaaccagc aaatcacagc ttcctctact caccgagctc tttttggact ccaaaaatgg
     1201 tatccctact atgcacgtct taataagaag gggcttataa atgcgtggac agctgcagaa
     1261 aatgacagat ggccgtggat tcagataaat ttgcaaagga aaatgagagt tactggtgtg
     1321 attacccaag gagccaagag gattggaagc ccagagtata taaaatccta caaaattgcc
     1381 tacagtaatg atggaaagac ttgggcaatg tacaaagtga aaggcaccaa tgaagacatg
     1441 gtgtttcgtg gaaacattga taacaacact ccatatgcta actctttcac accccccata
     1501 aaagctcagt atgtaagact ctatccccaa gtttgtcgaa gacattgcac tttgcgaatg
     1561 gaacttcttg gctgtgaact gtcgggttgt tctgagcctc tgggtatgaa atcaggacat
     1621 atacaagact atcagatcac tgcctccagc atcttcagaa cgctcaacat ggacatgttc
     1681 acttgggaac caaggaaagc tcggctggac aagcaaggca aagtgaatgc ctggacctct
     1741 ggccacaatg accagtcaca atggttacag gtggatcttc ttgttccaac caaagtgact
     1801 ggcatcatta cacaaggagc taaagatttt ggtcatgtac agtttgttgg ctcctacaaa
     1861 ctggcttaca gcaatgatgg agaacactgg actgtatacc aggatgaaaa gcaaagaaaa
     1921 gataaggttt tccagggaaa ttttgacaat gacactcaca gaaaaaatgt catcgaccct
     1981 cccatctatg cacgacacat aagaatcctt ccttggtcct ggtacgggag gatcacattg
     2041 cggtcagagc tgctgggctg cacagaggag gaagcggatc caccggtcgc caccatggtg
     2101 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2161 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2221 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2281 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     2341 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2401 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2461 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2521 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2581 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2641 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2701 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2761 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc
     2821 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     2881 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     2941 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3001 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     3061 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3121 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3181 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3241 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3301 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3361 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3421 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3481 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3541 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3601 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     3661 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3721 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3781 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3841 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3901 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3961 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     4021 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4081 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4141 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4201 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     4261 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4321 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4381 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4441 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4501 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4561 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4621 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4681 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4741 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4801 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4861 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4921 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4981 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     5041 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     5101 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     5161 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     5221 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     5281 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5341 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5401 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5461 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5521 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5581 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5641 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5701 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5761 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5821 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5881 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5941 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6001 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6061 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6121 ctgtggataa ccgtattacc gccatgcat
//
