LOCUS       sequence-84086-e        6725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human TRIM65 mammalian expression vector with 3xFLAG epitope at 
            c-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6725)
  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 6725)
  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: pCMVhTRIM65_3Flag
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     525..545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        620..638
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2233..2304
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(2357..2376)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2358..2376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2405..2422)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2411..2618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2625..3080)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2762..2783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2974..2993
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3107..3211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3212..3280,3281..4069)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3430..3449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        4163..4308
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     CDS             4351..5388
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     primer_bind     complement(5386..5405)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5430..5478
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6029..6617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6518..6537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      481 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      541 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      601 gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gctcgagctg
      661 aagcttgcca tggccgcgca gctgctggag gagaagctga cctgcgccat ctgcctgggg
      721 ctctaccagg acccagtgac gctgccctgc ggccacaact tctgcggggc ctgcatccgg
      781 gactggtggg accgctgcgg aaaggcgtgc cccgagtgcc gggagccctt tcccgacggc
      841 gccgagctgc gccgcaacgt ggccctcagc ggcgtgctgg aggtggtgcg cgccgggccc
      901 gcccgggatc ccggccccga tcccggcccc ggccccgacc ctgccgcgcg ctgcccccgc
      961 cacgggcggc cgctggagct cttctgccgg accgagggcc gctgtgtgtg cagcgtgtgc
     1021 accgtgcgcg agtgtcgcct ccacgagcgg gcgctgctgg atgccgagcg cctcaagcgc
     1081 gaggcccagc tgagagccag cctggaggtt acccagcagc aggccaccca ggccgaaggc
     1141 cagctactag agctgcgcaa gcaaagcagc cagatccaga actcggcctg catcttggcc
     1201 tcctgggtct ccggcaagtt cagcagcctg ctacaggccc tggaaataca gcacacgaca
     1261 gcactgagga gcatcgaggt ggccaagacg caggcgctgg cacaggctcg agacgaggag
     1321 cagcggctgc ggggccactt ggaggctgtg gctcgccatg gctgcaggat ccgggagctc
     1381 ctggagcagg tggatgagca gaccttcctg caggaatcgc agctcctcca gcccccaggg
     1441 cctcttgggc cactgacccc tctgcagtgg gatgaagacc aacagctggg tgacctgaag
     1501 cagttgctaa gccggctgtg tggcctcctc ttggaagagg ggagccaccc tggggcacca
     1561 gccaagcctg tggacttagc ccccgtggag gccccaggtc ccctggcacc ggtcccaagc
     1621 acagtttgtc cactgaggag gaaactctgg cagaattatc gcaatctgac ctttgatcca
     1681 gtcagcgcca accgtcactt ctatctgtcg cgccaggacc agcaggtgaa gcactgtcgt
     1741 cagtcccggg gcccaggcgg gcccggcagc tttgagctct ggcaggtgca atgtgcccag
     1801 agcttccagg ccgggcacca ctactgggag gtgcgcgcgt cagaccactc ggtgacactg
     1861 ggcgtctcct acccgcaact gccacggtgc aggctggggc cccacacaga caacattggc
     1921 cggggaccct gctcctgggg gctctgcgtc caggaggaca gcctccaggc ctggcacaac
     1981 ggggaagccc agcgcctccc aggggtgtca gggcggctcc tgggcatgga tttggacctg
     2041 gcctcaggct gcctcacctt ctacagcctg gagccccaga cccagcccct gtacaccttc
     2101 catgccctct tcaaccagcc cctcaccccc gtcttctggc tcctcgaggg taggaccctg
     2161 accctgtgcc atcagccagg ggctgtgttc cctccggggc cccaggaaga ggtgctcagc
     2221 atcgatgtcg aggattacaa ggatgacgac gataaggact ataaggacga tgatgacaag
     2281 gactacaaag atgatgacga taaatagggc ccggtacctt aattaattaa ggtaccaggt
     2341 aagtgtaccc aattcgccct atagtgagtc gtattacaat tcactcgatc ggctcgctga
     2401 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2461 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2521 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2581 ggggaggatt gggaagacaa tagcaggcat gctggggaac gcgtaaattg taagcgttaa
     2641 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2701 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2761 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2821 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2881 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2941 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3001 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3061 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3121 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3181 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3241 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3301 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3361 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3421 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3481 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3541 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3601 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3661 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3721 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3781 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3841 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3901 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3961 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4021 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggc ctcaggatta
     4081 gtgcttcatc cccgtggccc gttgctcgcg tttgctggcg gtgtccccgg aagaaatata
     4141 tttgcatgtc tttagttcta tgatgacaca aaccccgccc agcgtcttgt cattggcgaa
     4201 ttcgaacacg cagatgcagt cggggcggcg cggtccgagg tccacttcgc atattaaggt
     4261 gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca gcgtcaacag
     4321 cgtgccgcag atcccggggg gcaatgagat atgaaaaagc ctgaactcac cgcgacgtct
     4381 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     4441 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     4501 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     4561 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     4621 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     4681 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     4741 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     4801 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     4861 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     4921 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     4981 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     5041 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     5101 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     5161 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     5221 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     5281 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     5341 cgacgcccca gcactcgtcc ggatcgggag atgggggagg ctaactgaaa cacggaagga
     5401 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     5461 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     5521 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     5581 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     5641 tagccactgg ccccgtgggt tagggacggg gtcccccatg gggaatggtt tatggttcgt
     5701 gggggttatt attttgggcg ttgcgtgggg tcaggtccac gactggactg agcagacaga
     5761 cccatggttt ttggatggcc tgggcatgga ccgcatgtac tggcgcgaca cgaacaccgg
     5821 gcgtctgtgg ctgccaaaca cccccgaccc ccaaaaacca ccgcgcggat ttctggcgcc
     5881 gccggacgaa ctaaacctga ctacgacatc tctgcccctt cttcgctggt acgaggagcg
     5941 ctttgcctca ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6001 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6061 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6121 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6181 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6241 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6301 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6361 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6421 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6481 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6541 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6601 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6661 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6721 ccgcc
//
