LOCUS       sequence-258743-        8011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one huTRIM5 rescue-control shRNA knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8011)
  AUTHORS   Kim K, Dauphin A, Komurlu S, McCauley SM, Yurkovetskiy L, Carbone C,
            Diehl WE, Strambio-De-Castillia C, Campbell EM, Luban J
  TITLE     Cyclophilin A protects HIV-1 from restriction by human TRIM5alpha.
  JOURNAL   Nat Microbiol. 2019 Dec;4(12):2044-2051. doi: 
            10.1038/s41564-019-0592-5. Epub 2019 Oct 21.
  PUBMED    31636416
REFERENCE   2  (bases 1 to 8011)
  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: 
            "10.1038/s41564-019-0592-5"; journalName: "Nat Microbiol"; date: 
            "2019-12"; volume: "4"; issue: "12"; pages: "2044-2051"
COMMENT     Sequence Label: pPU-huTRIMmiR30-L1221
FEATURES             Location/Qualifiers
     source          1..8011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             324..920
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(324..343)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     820..840
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             921..1148
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-48 has been 
                     mutated to arginine"
                     /label=ubiquitin-K48R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     ncRNA           2716..2730
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     LTR             2864..3044
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3115..3703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3195..3214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3874..4665,4666..4734))
                     /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     4497..4516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4735..4839)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4914..4933)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5107..5127)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5112..5441
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5292..5427
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5354..5373
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        5453..5832
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5834..6032
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5987..6007
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6050..6230
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6277..6402
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6899..7132
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7317..7361
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7659..7776
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7913..8011,1..309)
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
ORIGIN
        1 ggatatctgc ggtgagcagt ttcggccccg gcccggggcc aagaacagat ggtcaccgca
       61 gtttcggccc cggcccgagg ccaagaacag atggtcccca gatatggccc aaccctcagc
      121 agtttcttaa gacccatcag atgtttccag gctcccccaa ggacctgaaa tgaccctgcg
      181 ccttatttga attaaccaat cagcctgctt ctcgcttctg ttcgcgcgct tctgcttccc
      241 gagctctata aaagagctca caacccctca ctcggcgcgc cagtcctccg acagactgag
      301 tcgcccgggg gtctagtgcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
      361 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
      421 cgcgccacac cgtggacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
      481 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
      541 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
      601 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
      661 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
      721 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
      781 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
      841 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
      901 tgacccgcaa gcccggtgcc atgcagatct ttgtgaagac cctcactggc aaaaccatca
      961 cccttgaggt cgagcccagt gacaccattg agaatgtcaa agccaaaatt caagacaagg
     1021 agggtatccc acctgaccag cagcgtctga tatttgccgg cagacagctg gaggatggcc
     1081 gcactctctc agactacaac atccagaaag agtccaccct gcacctggtg ttgcgcctgc
     1141 gaggtggcat ggcttctggg atcctggtta atgtaaagga ggaggtgacc tgccccatct
     1201 gcctggaact cctgacacaa cccctgagcc tggactgcgg ccacagcttc tgtcaggcct
     1261 gtttaacagc taaccacaag aagtccatgc tagacaaagg agagagtagc tgccctgtgt
     1321 gccggatcag ttaccagcct gagaacatac ggcctaatcg gcatgtagcc aacatagtgg
     1381 agaagctcag ggaggtcaag ttgagcccag aggggcagaa agttgatcat tgtgcacgcc
     1441 atggagagaa acttctactc ttctgtcagg aggacgggaa ggtcatttgc tggctttgtg
     1501 agcggtctca ggagcaccgt ggtcaccaca cgttcctcac agaggaggtt gcccgggagt
     1561 accaagtgaa gctccaggca gctctggaga tgctgaggca gaagcagcag gaagctgaag
     1621 agttggaagc tgacatcaga gaagagaaag cttcctggaa gactcaaata cagtatgaca
     1681 aaaccaacgt cttggcagat tttgagcaac tgagagacat cctggactgg gaggagagca
     1741 atgagctgca aaacctggag aaggaggagg aagacattct gaaaagcctt acgaactctg
     1801 aaactgagat ggtgcagcag acccagtccc tgagagagct catctcagat ctggagcatc
     1861 ggctgcaggg gtcagtgatg gagctgcttc agggtgtgga tggcgtcata aaaaggacgg
     1921 agaacgtgac cttgaagaag ccagaaactt ttccaaaaaa tcaaaggaga gtgtttcgag
     1981 ctcctgatct gaaaggaatg ctagaagtgt ttagagagct gacagatgtc cgacgctact
     2041 gggttgatgt gacagtggct ccaaacaaca tttcatgtgc tgtcatttct gaagataaga
     2101 gacaagtgag ctctccgaaa ccacagataa tatatggggc acgagggaca agataccaga
     2161 catttgtgaa tttcaattat tgtactggca tcctgggctc tcaaagtatc acatcaggga
     2221 aacattactg ggaggtagac gtgtccaaga aaactgcttg gatactgggg gtatgtgctg
     2281 gcttccaacc tgatgcaatg tgtaatattg aaaaaaatga aaattatcaa cctaaatacg
     2341 gctactgggt tatagggtta gaggaaggag ttaaatgtag tgctttccag gatagttcct
     2401 tccatactcc ttctgttcct ttcattgtgc ccctctctgt gattatttgt cctgatcgtg
     2461 ttggagtttt cctagactat gaggcttgca ctgtctcatt cttcaatatc acaaaccatg
     2521 gatttctcat ctataagttt tctcactgtt ctttttctca gcctgtattt ccatatttaa
     2581 atcctagaaa atgtggagtc cccatgactc tgtgctcacc aagctcttga gcggccgcct
     2641 tcttaaccca acagaaggct cgagaaggta tattgctgtt gacagtgagc gcacaaacgc
     2701 tctcatcgac aagtagtgaa gccacagatg tacttgtcga tgagagcgtt tgtatgccta
     2761 ctgcctcgga cttcaagggg ctagaattcg gcagctgtag atcttagcca ctttttaaaa
     2821 gaaaaggggg gactggaagg gctaactgca tccggactgt actgggtctc tctggttaga
     2881 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     2941 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     3001 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttcatgtg
     3061 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3121 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3181 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3241 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3301 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3361 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3421 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3481 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3541 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3601 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3661 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3721 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3781 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3841 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3901 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3961 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4021 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4081 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4141 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4201 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4261 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4321 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4381 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4441 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4501 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4561 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4621 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4681 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4741 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4801 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4861 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     4921 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     4981 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     5041 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     5101 cgctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     5161 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     5221 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     5281 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5341 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     5401 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcgcg ttgacattga
     5461 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     5521 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     5581 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     5641 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     5701 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     5761 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     5821 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     5881 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     5941 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     6001 aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg
     6061 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     6121 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     6181 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
     6241 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
     6301 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
     6361 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
     6421 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
     6481 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
     6541 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
     6601 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     6661 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     6721 aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga
     6781 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     6841 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     6901 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     6961 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     7021 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     7081 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     7141 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     7201 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     7261 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     7321 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     7381 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     7441 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     7501 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     7561 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
     7621 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     7681 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     7741 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     7801 gatccagttt ggttaattaa ctgcagcccc gataaaataa aagattttat ttagtctcca
     7861 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctg cagtaacgcc
     7921 attttgcaag gcatggaaaa ataccaaacc aagaatagag aagttcagat caagggcggg
     7981 tacatgaaaa tagctaacgt tgggccaaac a
//
