LOCUS       sequence-492422-        6140 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  mammalian expression construct for human ectonucleotide 
            pyrophosphatase/phosphodiesterase family member 5 C-terminally 
            tagged with EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  TITLE     GFP-tagged lysosomal proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6140)
  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            "Unpublished"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: hENPP5-EGFP
FEATURES             Location/Qualifiers
     source          1..6140
                     /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      2080..2089
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2086..2088,2089..2091,2092..2805)
                     /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(2086..2088,2089..2091,2092..2802)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2131..2152)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2392..2411)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2739..2760
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2928..3049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2965..2984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3019..3038
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3056..3511)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3193..3214)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3405..3424
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3538..3642
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3644..4001
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3644..3835
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3667..3687)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3852..3987
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4036..4830
                     /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             4036..4830
                     /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(4090..4109)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4700..4719
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5018..5037)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5062..5109
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5438..6026
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5927..5946
                     /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 accatgactt cgaaatttct cttggtgtcc
      661 ttcatacttg ctgcactgag tctttcaacc accttttctc tccaaccaga ccagcaaaag
      721 gttctactag tttcttttga tggattccgt tgggattact tatataaagt tccaacgccc
      781 cattttcatt atattatgaa atatggtgtt cacgtgaagc aagttactaa tgtttttatt
      841 acaaaaacct accctaacca ttatactttg gtaactggcc tctttgcaga gaatcatggg
      901 attgttgcaa atgatatgtt tgatcctatt cggaacaaat ctttctcctt ggatcacatg
      961 aatatttatg attccaagtt ttgggaagaa gcgacaccaa tatggatcac aaaccagagg
     1021 gcaggacata ctagtggtgc agccatgtgg cccggaacag atgtaaaaat acataagcgc
     1081 tttcctactc attacatgcc ttacaatgag tcagtttcat ttgaagatag agttgccaaa
     1141 attattgaat ggtttacgtc aaaagagccc ataaatcttg gtcttctcta ttgggaagac
     1201 cctgatgaca tgggccacca tttgggacct gacagtccgc tcatggggcc tgtcatttca
     1261 gatattgaca agaagttagg atatctcata caaatgctga aaaaggcaaa gttgtggaac
     1321 actctgaacc taatcatcac aagtgatcat ggaatgacgc agtgctctga ggaaaggtta
     1381 atagaacttg accagtacct ggataaagac cactataccc tgattgatca atctccagta
     1441 gcagccatct tgccaaaaga aggtaaattt gatgaagtct atgaagcact aactcacgct
     1501 catcctaatc ttactgttta caaaaaagaa gacgttccag aaaggtggca ttacaaatac
     1561 aacagtcgaa ttcaaccaat catagcagtg gctgatgaag ggtggcacat tttacagaat
     1621 aagtcagatg actttctgtt aggcaaccac ggttacgata atgcgttagc agatatgcat
     1681 ccaatatttt tagcccatgg tcctgccttc agaaagaatt tctcaaaaga agccatgaac
     1741 tccacagatt tgtacccact actatgccac ctcctcaata tcaccgccat gccacacaat
     1801 ggatcattct ggaatgtcca ggatctgctc aattcagcaa tgccaagggt ggtcccttat
     1861 acacagagta ctatactcct ccctggtagt gttaaaccag cagaatatga ccaagagggg
     1921 tcataccctt atttcatagg ggtctctctt ggcagcatta tagtgattgt attttttgta
     1981 attttcatta agcatttaat tcacagtcaa atacctgcct tacaagatat gcatgctgaa
     2041 atagctcaac cattattaca agccgcggat ccaccggtcg ccaccatggt gagcaagggc
     2101 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2161 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2221 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2281 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2341 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2401 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2461 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2521 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2581 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2641 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     2701 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     2761 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct
     2821 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2881 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2941 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3001 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     3061 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3121 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3181 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3241 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3301 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3361 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3421 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3481 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     3541 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3601 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     3661 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3721 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3781 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3841 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3901 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3961 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     4021 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4081 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4141 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4201 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4261 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4321 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4381 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4441 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4501 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4561 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4621 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4681 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4741 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4801 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4861 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4921 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4981 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     5041 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     5101 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     5161 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     5221 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     5281 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5341 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5401 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5461 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5521 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5581 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5641 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5701 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5761 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5821 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5881 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5941 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6001 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6061 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6121 accgtattac cgccatgcat
//
