LOCUS       sequence-273548-        6069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Eukaryotic expression of rat GRASP65 full-length.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6069)
  TITLE     Yanzhuang Wang Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6069)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pEGFP-N2-GRASP65
FEATURES             Location/Qualifiers
     source          1..6069
                     /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      2009..2018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2015..2017,2018..2020,2021..2734)
                     /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(2015..2017,2018..2020,2021..2731)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2060..2081)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2321..2340)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2668..2689
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2857..2978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2894..2913)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2948..2967
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2985..3440)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3122..3143)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3334..3353
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3467..3571
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3573..3930
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3573..3764
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3596..3616)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3781..3916
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3965..4759
                     /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             3965..4759
                     /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(4019..4038)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4629..4648
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4947..4966)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4991..5038
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5367..5955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5856..5875
                     /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 caagcttcga attcatgggg ctaggggcaa gcagcgagca
      661 gccggcgggc ggcgagggct tccatctgca cggggtacaa gagaactcgc cagcccagca
      721 ggcgggcctg gagccctact ttgacttcat catcaccatc gggcactcaa ggctgaacaa
      781 ggagaatgac acgctgaagg cactgctgaa ggctaatgtg gagaagccgg tgaagctgga
      841 ggtgttcaac atgaagacca tgagggtgcg agaggtggag gtggtgccca gcaacatgtg
      901 gggcggccag ggcctgctgg gagctagcgt gcgcttctgc agcttccgca gggccagcga
      961 gcacgtgtgg catgtgctgg atgtggagcc ctcttcaccc gctgccttgg ccggcctgcg
     1021 cccttacaca gactacattg ttggctctga ccagatcctc caggagtcgg aagacttctt
     1081 cactctcatt gagtcccatg agggaaagcc cctgaagctg atggtttata actctgaatc
     1141 cgactcctgc cgggaggtga ctgtgacccc caatgcagcc tggggtggag agggcagtct
     1201 ggggtgtggt attggttacg ggtacctgca ccggatccca acgcagccct ccagccagta
     1261 caagaagcca cccagtgcct catcacctgg cactccagct aagaccccac aacctaatgc
     1321 ctttcctctt ggtgccccac caccttggcc catccctcag gactcttctg gcccagagct
     1381 gggttccagg cagagtgact acatggaggc cttaccacag gtccctggtg gctttatgga
     1441 ggagcagctc cctgggcctg ggagtcctgg ccatggcact gctgactatg ggggatgcct
     1501 acattccatg gagatcccgc ttcagcctcc acctccagtc cagcgggtca tggacccagg
     1561 cttcctggat gtgtcaggca tgtccctcct ggacagcagc aatacaagtg tgtgccccag
     1621 cttgtcatct tcctcattgc tgacccctac agctgtttca gccttaggac cagaggacat
     1681 tggttccagc agcagttctc atgagcgggg tggtgaggcc acgtggtcag ggtcagagtt
     1741 cgagatctcc ttccccgaca gtccaggctc ccaggcccag gtggaccacc tgccccggct
     1801 gactctccct gatggcctca cgtctgcagc ttcaccagaa gaagggctgt ctgcagaact
     1861 gctggaagca cagaccgagg aaccagcaga cacagccagc ctggattgca tggcagagac
     1921 tgaggggcca gccggccaag tccaggctgc cccagatcca gagcctgggt tgtgtgaagg
     1981 cccctggccc gggatccacc ggccggtcgc caccatggtg agcaagggcg aggagctgtt
     2041 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2101 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2161 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2221 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2281 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2341 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2401 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2461 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2521 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2581 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2641 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2701 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc
     2761 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     2821 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2881 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2941 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     3001 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     3061 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     3121 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     3181 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     3241 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     3301 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3361 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3421 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3481 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3541 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     3601 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3661 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3721 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3781 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3841 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3901 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     3961 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4021 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4081 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4141 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4201 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4261 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4321 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4381 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4441 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     4501 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4561 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4621 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4681 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4741 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4801 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4861 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4921 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4981 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     5041 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     5101 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     5161 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     5221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6001 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6061 gccatgcat
//
