LOCUS       sequence-409130-        9021 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 9021)
  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 9021)
  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: LCAPCW
FEATURES             Location/Qualifiers
     source          1..9021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     enhancer        2201..2504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2505..2708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2659..2679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2705..2729
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3877..4376
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3912..3935)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4309..4328
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4394..5104
                     /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(4539..4557)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5165..5185)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5583..5594)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5784..6417
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6546..6562)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6546..6562)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6559..6581)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6570..6586
                     /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(6594..6600,6601..6618,6619..6624))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6639..6660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6777..6794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6948..7536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7028..7047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7707..8498,8499..8567))
                     /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     8330..8349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8568..8672)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8720..8854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8757..8776)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8811..8830
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa
     2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     2761 tccatagaag acaccgactc tactagagga tcgctagcgc ttcgactagg gataacaggg
     2821 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     2881 cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt atattgctgt
     2941 tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt gttgctctca
     3001 atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa ttcaagtttc
     3061 aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag tccactaatg
     3121 ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa ccttgtattc
     3181 catctttatc tctagctgag tcatcaatta catatccata actttcttca taagcaaaaa
     3241 caaaatttaa tccgttatct tcttctttag caatttctct acccattcat ttcaatccag
     3301 ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca cccaaatcac
     3361 ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag cgttttagat
     3421 ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat cttacaaaat
     3481 gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc ataataataa
     3541 tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga tcaaattctg
     3601 gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct tcaccctcaa
     3661 taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct gttccattaa
     3721 ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga gcaattatct
     3781 tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa
     3841 aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg cttttcccaa
     3901 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     3961 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     4021 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     4081 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     4141 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     4201 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     4261 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     4321 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     4381 ttaattaacc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     4441 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     4501 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     4561 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     4621 atacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     4681 caactgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     4741 ctccctacag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     4801 cgacggcccc gtaatgcagt gtcgtacaat gggctgggag gcttccactg agcggatgta
     4861 ccccgaggac ggtgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     4921 ccactacgac gctgaagtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     4981 agcctacaac gtggacatca agttggacat cctttcccac aacgaggact acaccatcgt
     5041 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     5101 gtaggtttaa acatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     5161 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     5221 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     5281 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     5341 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     5401 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     5461 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     5521 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5581 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5641 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     5701 ctggcctgca ggggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     5761 ctttttaaaa gaaaagaggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     5821 ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga actacacacc
     5881 agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     5941 gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag
     6001 cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct
     6061 agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg
     6121 agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac
     6181 tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt
     6241 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     6301 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     6361 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     6421 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     6481 gtgagaggcc ttgacattgc tagcgtttta ccgtcgacct ctagctagag cttggcgtaa
     6541 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     6601 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     6661 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6721 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6781 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6841 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6901 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6961 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     7021 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     7081 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     7141 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     7201 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     7261 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     7321 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7381 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7441 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7501 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7561 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7621 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7681 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7741 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7801 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7861 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7921 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7981 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     8041 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     8101 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     8161 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     8221 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     8281 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     8341 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8401 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8461 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8521 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8581 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8641 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     8701 gtcgacggat cgggagatca acttgtttat tgcagcttat aatggttaca aataaagcaa
     8761 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     8821 caaactcatc aatgtatctt atcatgtctg gatcaactgg ataactcaag ctaaccaaaa
     8881 tcatcccaaa cttcccaccc cataccctat taccactgcc aattacctgt ggtttcattt
     8941 actctaaacc tgtgattcct ctgaattatt ttcattttaa agaaattgta tttgttaaat
     9001 atgtactaca aacttagtag t
//
