LOCUS       sequence-430327-        8988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with E2-Crimson reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8988)
  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 8988)
  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: LCAPEW
FEATURES             Location/Qualifiers
     source          1..8988
                     /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)"
     enhancer        2200..2503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2504..2707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2658..2678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2704..2728
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3876..4375
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3911..3934)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4308..4327
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4393..5070
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(5131..5151)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5549..5560)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5750..6383
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6512..6528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6512..6528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6525..6547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6536..6552
                     /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(6560..6566,6567..6584,6585..6590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6605..6626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6743..6760)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6914..7502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6994..7013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7673..8464,8465..8533))
                     /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     8296..8315
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8534..8638)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8686..8820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8723..8742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8777..8796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(8988,1..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 gataagcttg ggagttccgc gttacataac ttacggtaaa
     2221 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     2281 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2341 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2401 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2461 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2521 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2581 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2641 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2701 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
     2761 ccatagaaga caccgactct actagaggat cgctagcgct tcgactaggg ataacagggt
     2821 aattgtttga atgaggcttc agtactttac agaatcgttg cctgcacatc ttggaaacac
     2881 ttgctgggat tacttcgact tcttaaccca acagaaggct cgagaaggta tattgctgtt
     2941 gacaggatcc acattaacaa ctttaagtcc agcaatttga gttaagggtg ttgctctcaa
     3001 tgatttcact aatggttcaa tttttaattt cttttcttct ggttcaaaat tcaagtttca
     3061 agtgaaagtg taatatgcac ccatttcttt caataaatct tctaaatagt ccactaatgt
     3121 tttattttgt tttttacaaa atcaagcagc ctctgctatc aatatagaac cttgtattcc
     3181 atctttatct ctagctgagt catcaattac atatccataa ctttcttcat aagcaaaaac
     3241 aaaatttaat ccgttatctt cttctttagc aatttctcta cccattcatt tcaatccagt
     3301 taaagttttt acaatcttaa ctccatattt ttcctgagcg attctatcac ccaaatcact
     3361 tgttacaaaa cttgaatata gagccggatt ttttggaatg ctatttaagc gttttagatt
     3421 tgataatttt caatcaatta aaattggtcc tgtttgattt ccatctaatc ttacaaaatg
     3481 accatcatgt tttattgcca ttccaaatct gtcagcatct gggtcattca taataataat
     3541 atctgcatca tgtttaatac catattcaag cggtattttt catgcaggat caaattctgg
     3601 atttggattt acaacatttt tcaatgtttc atcttcaaat gcacgatctt caccctcaat
     3661 aacgttatat cctgattcac gtaatctttt tggggtaaat ttagttcctg ttccattaac
     3721 tgcgctaaaa atagggccct gcctcggact tcaaggggct agaattcgag caattatctt
     3781 gtttactaaa actgaatacc ttgctatctc tttgatacat ttttacaaag ctgaattaaa
     3841 atggtataaa ttaaatcact tttttcaatt ctaccgggta ggggaggcgc ttttcccaag
     3901 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     3961 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     4021 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     4081 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     4141 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     4201 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     4261 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     4321 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctt
     4381 taattaacca ccatggatag cactgagaac gtcatcaagc ccttcatgcg cttcaaggtg
     4441 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgtggg cgagggcaag
     4501 ccctacgagg gcacccagac cgccaagctg caagtgacca agggcggccc cctgcccttc
     4561 gcctgggaca tcctgtcccc ccagttcttc tacggctcca aggcgtacat caagcacccc
     4621 gccgacatcc ccgactacct caagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     4681 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcatccct gcaagacggc
     4741 accctcatct accacgtcaa gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg
     4801 cagaagaaaa ctcttggctg ggagccctcc actgagcgca actacccccg cgacggcgtg
     4861 ctgaagggcg agaaccacat ggcgctgaag ctgaagggcg gcggccacta cctgtgtgag
     4921 ttcaagtcca tctacatggc taagaagccc gtgaagctgc ccggctacca ctacgtggac
     4981 tacaagctcg acatcacctc ccacaacgag gactacaccg tggtggagca gtacgaacgc
     5041 gccgaggccc gccaccacct gttccagtag gtttaaacat caacctctgg attacaaaat
     5101 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     5161 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     5221 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     5281 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     5341 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     5401 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5461 gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct
     5521 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5581 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5641 gatctccctt tgggccgcct ccccgcctgg cctgcagggg tacctttaag accaatgact
     5701 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa agaggggact ggaagggcta
     5761 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     5821 ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga
     5881 tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag
     5941 aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg
     6001 ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg
     6061 gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt
     6121 ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa
     6181 gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6241 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6301 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6361 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     6421 acttgcaaag aaatgaatat cagagagtga gaggccttga cattgctagc gttttaccgt
     6481 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6541 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6601 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6661 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6721 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6781 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6841 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6901 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6961 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7021 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7081 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7141 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7201 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7261 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7321 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7381 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7441 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7501 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7561 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7621 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7681 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7741 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7801 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7861 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7921 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7981 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8041 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8101 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8161 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8221 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8281 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8341 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8401 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8461 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8521 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8581 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8641 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcaactt gtttattgca
     8701 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     8761 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     8821 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc
     8881 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca
     8941 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtt
//
