LOCUS       sequence-409399-        8889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with mCherry reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8889)
  AUTHORS   Wang D, Xiu J, Zhao J, Luo J
  TITLE     miR-AB, a miRNA-based shRNA viral toolkit for multicolor-barcoded 
            multiplex RNAi at a single-cell level.
  JOURNAL   EMBO Rep. 2022 Apr 5;23(4):e53691. doi: 10.15252/embr.202153691. 
            Epub 2022 Feb 24.
  PUBMED    35201651
REFERENCE   2  (bases 1 to 8889)
  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: "EMBO Rep.";
            date: "2022-04-5"; volume: "23(4):e53691. doi"; pages: " 
            10.15252/embr.202153691. Epub 2022 Feb 24"
COMMENT     Sequence Label: LHAPCW
FEATURES             Location/Qualifiers
     source          1..8889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    680..805
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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    2027..2142
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2184..2395
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        3744..4243
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3779..3802)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4176..4195
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4261..4971
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4406..4424)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5032..5052)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5450..5461)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5651..6284
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6413..6429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6413..6429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6426..6448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6437..6453
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6461..6467,6468..6485,6486..6491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6506..6527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6644..6661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6815..7403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6895..6914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7574..8365,8366..8434))
                     /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     8197..8216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8435..8539)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8587..8721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8624..8643)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8678..8697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(8889,1..633)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac
       61 acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact
      121 gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa
      181 taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga
      241 gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga
      301 gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc
      361 tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc
      421 ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag
      481 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      541 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      601 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      661 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      721 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      781 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      841 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      901 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      961 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1021 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1081 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1141 agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1981 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccttta aaagaaaagg
     2041 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2101 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2161 cagcagagat ccagtttatc gatgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2221 ggggggaggg gtcggcaatt gaaccggagc ctagagaagg tggcgcgggg taaactggga
     2281 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2341 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagggctc
     2401 gcatctctcc ttcacgcgcc cgccgcctta cctgaggccg ccatccacgc cggttgagtc
     2461 gcgttctgcc gcctcccgcc tgtggtgcct cctgaactac gtccgccgtc taggtaagtt
     2521 tagagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca
     2581 gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt
     2641 ctgttctgcg ccgttacaga tcagcgcttc gactagggat aacagggtaa ttgtttgaat
     2701 gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta
     2761 cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga caggatccac
     2821 attaacaact ttaagtccag caatttgagt taagggtgtt gctctcaatg atttcactaa
     2881 tggttcaatt tttaatttct tttcttctgg ttcaaaattc aagtttcaag tgaaagtgta
     2941 atatgcaccc atttctttca ataaatcttc taaatagtcc actaatgttt tattttgttt
     3001 tttacaaaat caagcagcct ctgctatcaa tatagaacct tgtattccat ctttatctct
     3061 agctgagtca tcaattacat atccataact ttcttcataa gcaaaaacaa aatttaatcc
     3121 gttatcttct tctttagcaa tttctctacc cattcatttc aatccagtta aagtttttac
     3181 aatcttaact ccatattttt cctgagcgat tctatcaccc aaatcacttg ttacaaaact
     3241 tgaatataga gccggatttt ttggaatgct atttaagcgt tttagatttg ataattttca
     3301 atcaattaaa attggtcctg tttgatttcc atctaatctt acaaaatgac catcatgttt
     3361 tattgccatt ccaaatctgt cagcatctgg gtcattcata ataataatat ctgcatcatg
     3421 tttaatacca tattcaagcg gtatttttca tgcaggatca aattctggat ttggatttac
     3481 aacatttttc aatgtttcat cttcaaatgc acgatcttca ccctcaataa cgttatatcc
     3541 tgattcacgt aatctttttg gggtaaattt agttcctgtt ccattaactg cgctaaaaat
     3601 agggccctgc ctcggacttc aaggggctag aattcgagca attatcttgt ttactaaaac
     3661 tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaatt
     3721 aaatcacttt tttcaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3781 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     3841 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     3901 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     3961 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     4021 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     4081 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     4141 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     4201 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgaccttta attaaccacc
     4261 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     4321 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     4381 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     4441 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggcata cgtgaagcac
     4501 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa ctgggagcgc
     4561 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctacaggac
     4621 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4681 atgcagtgtc gtacaatggg ctgggaggct tccactgagc ggatgtaccc cgaggacggt
     4741 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     4801 gaagtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggagc ctacaacgtg
     4861 gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga acagtacgaa
     4921 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta ggtttaaaca
     4981 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     5041 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     5101 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     5161 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     5221 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     5281 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     5341 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     5401 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     5461 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     5521 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gcctgcaggg
     5581 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     5641 aagaggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg
     5701 atctaccaca cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc
     5761 agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta
     5821 gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg
     5881 gatgacccgg agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac
     5941 gtggcccgag agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag
     6001 ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga
     6061 gccctcagat cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga
     6121 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6181 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6241 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca
     6301 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggccttg
     6361 acattgctag cgttttaccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     6421 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     6481 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     6541 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6601 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6661 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6721 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6781 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6841 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6901 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6961 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     7021 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     7081 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     7141 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     7201 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     7261 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7321 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7381 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7441 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7501 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7561 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7621 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7681 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7741 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7801 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7861 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7921 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7981 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     8041 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     8101 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     8161 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     8221 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     8281 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     8341 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     8401 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     8461 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     8521 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     8581 gagatcaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     8641 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     8701 gtatcttatc atgtctggat caactggata actcaagcta accaaaatca tcccaaactt
     8761 cccaccccat accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt
     8821 gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac
     8881 ttagtagtt
//
