LOCUS       40924_29986        6083 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of m-Cherry-tagged wildtype mouse Trim21 in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  AUTHORS   Clift D, McEwan WA, Labzin LI, Konieczny V, Mogessie B, James LC, 
            Schuh M
  TITLE     A Method for the Acute and Rapid Degradation of Endogenous Proteins.
  JOURNAL   Cell. 2017 Nov 13. pii: S0092-8674(17)31255-2. doi: 
            10.1016/j.cell.2017.10.033.
  PUBMED    29153837
REFERENCE   2  (bases 1 to 6083)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6083)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2017 
            Nov 13. pii: S0092-8674(17)31255-2. doi: 
            10.1016/j.cell.2017.10.033."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6083
                     /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"
     CDS             613..1320
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             1357..2742
                     /codon_start=1
                     /label=TRIM21
                     /note="mouse RING E3 ubiquitin ligase that can be used
                     together with antibodies to degrade intracellular proteins 
                     (Clift et al., 2018)"
                     /translation="MSLEKMWEEVTCSICLDPMVEPMSIECGHCFCKECIFEVGKNGGS
                     SCPECRQQFLLRNLRPNRHIANMVENLKQIAQNTKKSTQETHCMKHGEKLHLFCEEDGQ
                     ALCWVCAQSGKHRDHTRVPIEEAAKVYQEKIHVALEKLRKGKELAEKMEMDLTMQRTDW
                     KRNIDTQKSRIHAEFALQNSLLAQEEQRQLQRLEKDQREYLRLLGKKEAELAEKNQALQ
                     ELISELERRIRGSELELLQEVRIILERSGSWNLDTLDIDAPDLTSTCPVPGRKKMLRTC
                     WVHITLDRNTANSWLIISKDRRQVRMGDTHQNVSDNKERFSNYPMVLGAQRFSSGKMYW
                     EVDVTQKEAWDLGVCRDSVQRKGQFSLSPENGFWTIWLWQDSYEAGTSPQTTLHIQVPP
                     CQIGIFVDYEAGVVSFYNITDHGSLIYTFSECVFAGPLRPFFNVGFNYSGGNAAPLKLC
                     PLKM"
     polyA_signal    2871..2992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2999..3454)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3481..3585
                     /label=AmpR promoter
     promoter        3587..3944
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3979..4770
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4961..4980)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5005..5052
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5381..5969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tccggactca gatctcgagc tcaagcttcg aattcaatgt ctctggaaaa gatgtgggag
     1381 gaggtcacct gttctatctg cctggatccc atggtggagc ctatgagtat cgaatgtggc
     1441 cattgctttt gcaaggaatg catttttgaa gttgggaaga atgggggcag ttcatgtccc
     1501 gagtgccggc aacagtttct gctccgaaac ctcaggccca atagacatat agccaacatg
     1561 gtggaaaacc ttaaacagat agcccagaat accaagaaga gtacccagga aacgcactgc
     1621 atgaagcatg gagagaagct tcacctattc tgtgaggaag atgggcaggc cctttgctgg
     1681 gtgtgtgccc agtctgggaa acaccgggac cacaccaggg tccctattga agaggctgct
     1741 aaggtatacc aggagaagat ccacgtggct ttagaaaaac tgagaaaggg gaaagagttg
     1801 gccgagaaga tggaaatgga tctcacgatg caaagaacag actggaagag gaacattgac
     1861 acccagaagt cgaggattca cgcagagttc gcacttcaga atagcttgct ggctcaggag
     1921 gagcagaggc agctgcagag gctggagaag gatcaaaggg agtacctgag actcctgggg
     1981 aagaaggagg ctgagctggc tgagaagaac caggccctgc aggagctgat ctcagagctg
     2041 gagaggagga ttcgtggttc agagctggag ctactgcagg aggtgaggat catcctggaa
     2101 aggagtggat cctggaacct ggacacgtta gatattgacg ccccagacct aacaagcaca
     2161 tgccctgtgc cagggcggaa gaagatgctg aggacgtgtt gggttcatat tactctggat
     2221 cgcaacaccg ccaactcatg gctcatcatc tcaaaggatc ggagacaagt gaggatggga
     2281 gacacccatc agaacgtgtc tgacaataag gagaggttta gtaattaccc catggtgcta
     2341 ggtgcccaga gattctcctc tgggaagatg tactgggagg tagatgtgac tcaaaaggag
     2401 gcctgggatc tgggggtttg cagagattct gttcagagga aagggcagtt ttcactcagt
     2461 cccgagaatg gcttctggac catttggtta tggcaagaca gctatgaggc tggtaccagt
     2521 cctcagacca ccctccacat tcaagtacct ccatgccaaa ttgggatctt tgtggactat
     2581 gaggctggcg ttgtctcctt ctacaacata actgaccatg gctccctcat ttacaccttc
     2641 tcggagtgtg tttttgctgg acctctgcga cctttcttca atgttggttt caattatagt
     2701 gggggaaatg cagcgcctct aaagctctgt ccactaaaga tgtgagcggc cgctctagat
     2761 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     2821 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     2881 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2941 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa
     3001 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3061 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3121 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3181 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3241 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3301 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3361 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3421 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     3481 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3541 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     3601 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3661 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3721 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3781 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3841 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     3901 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     3961 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4021 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4081 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4141 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     4201 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4261 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4321 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4381 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4441 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4501 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4561 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4621 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4681 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4741 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4801 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4861 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4921 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     4981 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     5041 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     5101 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     5161 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     5221 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5281 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5341 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5401 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5461 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5521 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5581 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5641 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5701 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5761 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5821 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5881 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5941 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6001 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6061 ataaccgtat taccgccatg cat
//
