LOCUS       sequence-84446-e        6281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for human TRIM65 fusion protein with 
            c-terminal EGFP. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6281)
  AUTHORS   Li S, Wang L, Fu B, Berman MA, Diallo A, Dorf ME
  TITLE     TRIM65 regulates microRNA activity by ubiquitination of TNRC6.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 May 13;111(19):6970-5. doi: 
            10.1073/pnas.1322545111. Epub 2014 Apr 28.
  PUBMED    24778252
REFERENCE   2  (bases 1 to 6281)
  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"; doi: 
            "10.1073/pnas.1322545111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-05-13- 13"; volume: "111"; issue: "19"; pages: "6970-5"
COMMENT     Sequence Label: hTRIM65_EGFP_N1
FEATURES             Location/Qualifiers
     source          1..6281
                     /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      619..628
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2221..2230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2227..2229,2230..2232,2233..2946)
                     /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(2227..2229,2230..2232,2233..2943)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2272..2293)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2533..2552)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2880..2901
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3069..3190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3160..3179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3197..3652)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3334..3355)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3546..3565
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3679..3783
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3785..4142
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3785..3976
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3808..3828)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3993..4128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4177..4971
                     /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             4177..4971
                     /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(4231..4250)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4841..4860
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5159..5178)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5203..5250
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5579..6167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6068..6087
                     /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 atctcgacgc caccatggcc gcgcagctgc tggaggagaa gctgacctgc
      661 gccatctgcc tggggctcta ccaggaccca gtgacgctgc cctgcggcca caacttctgc
      721 ggggcctgca tccgggactg gtgggaccgc tgcggaaagg cgtgccccga gtgccgggag
      781 ccctttcccg acggcgccga gctgcgccgc aacgtggccc tcagcggcgt gctggaggtg
      841 gtgcgcgccg ggcccgcccg ggatcccggc cccgatcccg gccccggccc cgaccctgcc
      901 gcgcgctgcc cccgccacgg gcggccgctg gagctcttct gccggaccga gggccgctgt
      961 gtgtgcagcg tgtgcaccgt gcgcgagtgt cgcctccacg agcgggcgct gctggatgcc
     1021 gagcgcctca agcgcgaggc ccagctgaga gccagcctgg aggttaccca gcagcaggcc
     1081 acccaggccg aaggccagct actagagctg cgcaagcaaa gcagccagat ccagaactcg
     1141 gcctgcatct tggcctcctg ggtctccggc aagttcagca gcctgctaca ggccctggaa
     1201 atacagcaca cgacagcact gaggagcatc gaggtggcca agacgcaggc gctggcacag
     1261 gctcgagacg aggagcagcg gctgcggggc cacttggagg ctgtggctcg ccatggctgc
     1321 aggatccggg agctcctgga gcaggtggat gagcagacct tcctgcagga atcgcagctc
     1381 ctccagcccc cagggcctct tgggccactg acccctctgc agtgggatga agaccaacag
     1441 ctgggtgacc tgaagcagtt gctaagccgg ctgtgtggcc tcctcttgga agaggggagc
     1501 caccctgggg caccagccaa gcctgtggac ttagcccccg tggaggcccc aggtcccctg
     1561 gcaccggtcc caagcacagt ttgtccactg aggaggaaac tctggcagaa ttatcgcaat
     1621 ctgacctttg atccagtcag cgccaaccgt cacttctatc tgtcgcgcca ggaccagcag
     1681 gtgaagcact gtcgtcagtc ccggggccca ggcgggcccg gcagctttga gctctggcag
     1741 gtgcaatgtg cccagagctt ccaggccggg caccactact gggaggtgcg cgcgtcagac
     1801 cactcggtga cactgggcgt ctcctacccg caactgccac ggtgcaggct ggggccccac
     1861 acagacaaca ttggccgggg accctgctcc tgggggctct gcgtccagga ggacagcctc
     1921 caggcctggc acaacgggga agcccagcgc ctcccagggg tgtcagggcg gctcctgggc
     1981 atggatttgg acctggcctc aggctgcctc accttctaca gcctggagcc ccagacccag
     2041 cccctgtaca ccttccatgc cctcttcaac cagcccctca cccccgtctt ctggctcctc
     2101 gagggtagga ccctgaccct gtgccatcag ccaggggctg tgttccctcc ggggccccag
     2161 gaagaggtgc tcagccgaat tctgcagtcg acggtaccgc gggcccggga tccaccggtc
     2221 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     2281 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     2341 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     2401 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     2461 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2521 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2581 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2641 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     2701 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     2761 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2821 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2881 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2941 aagtaaagcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
     3001 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3061 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3121 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3181 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3241 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3301 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3361 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3421 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3481 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3541 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3601 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     3661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3721 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3781 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3841 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3901 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3961 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4021 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4081 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4141 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4201 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4261 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4321 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     4381 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4441 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4501 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4561 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4621 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4681 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     4741 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4801 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4861 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4921 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4981 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5041 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5101 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     5161 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     5221 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     5281 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     5341 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     5401 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     5461 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5521 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5581 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5641 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5701 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     5761 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5821 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5881 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5941 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6001 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6061 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6121 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6181 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6241 tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
