LOCUS       sequence-409028-        8856 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 8856)
  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 8856)
  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: LHAPEW
FEATURES             Location/Qualifiers
     source          1..8856
                     /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)"
     promoter        2185..2396
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        3745..4244
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3780..3803)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4177..4196
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4262..4939
                     /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(5000..5020)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5418..5429)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5619..6252
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6381..6397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6381..6397)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6394..6416)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6405..6421
                     /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(6429..6435,6436..6453,6454..6459))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6474..6495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6612..6629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6783..7371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6863..6882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7542..8333,8334..8402))
                     /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     8165..8184
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8403..8507)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8555..8689
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8592..8611)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8646..8665
                     /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 cgatgggcag agcgcacatc gcccacagtc cccgagaagt
     2221 tggggggagg ggtcggcaat tgaaccggag cctagagaag gtggcgcggg gtaaactggg
     2281 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2341 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagggct
     2401 cgcatctctc cttcacgcgc ccgccgcctt acctgaggcc gccatccacg ccggttgagt
     2461 cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaacta cgtccgccgt ctaggtaagt
     2521 ttagagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc
     2581 agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt
     2641 tctgttctgc gccgttacag atcagcgctt cgactaggga taacagggta attgtttgaa
     2701 tgaggcttca gtactttaca gaatcgttgc ctgcacatct tggaaacact tgctgggatt
     2761 acttcgactt cttaacccaa cagaaggctc gagaaggtat attgctgttg acaggatcca
     2821 cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat gatttcacta
     2881 atggttcaat ttttaatttc ttttcttctg gttcaaaatt caagtttcaa gtgaaagtgt
     2941 aatatgcacc catttctttc aataaatctt ctaaatagtc cactaatgtt ttattttgtt
     3001 ttttacaaaa tcaagcagcc tctgctatca atatagaacc ttgtattcca tctttatctc
     3061 tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca aaatttaatc
     3121 cgttatcttc ttctttagca atttctctac ccattcattt caatccagtt aaagttttta
     3181 caatcttaac tccatatttt tcctgagcga ttctatcacc caaatcactt gttacaaaac
     3241 ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt gataattttc
     3301 aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga ccatcatgtt
     3361 ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata tctgcatcat
     3421 gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga tttggattta
     3481 caacattttt caatgtttca tcttcaaatg cacgatcttc accctcaata acgttatatc
     3541 ctgattcacg taatcttttt ggggtaaatt tagttcctgt tccattaact gcgctaaaaa
     3601 tagggccctg cctcggactt caaggggcta gaattcgagc aattatcttg tttactaaaa
     3661 ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa tggtataaat
     3721 taaatcactt ttttcaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3781 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3841 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3901 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3961 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     4021 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     4081 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     4141 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     4201 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgaccttt aattaaccac
     4261 catggatagc actgagaacg tcatcaagcc cttcatgcgc ttcaaggtgc acatggaggg
     4321 ctccgtgaac ggccacgagt tcgagatcga gggcgtgggc gagggcaagc cctacgaggg
     4381 cacccagacc gccaagctgc aagtgaccaa gggcggcccc ctgcccttcg cctgggacat
     4441 cctgtccccc cagttcttct acggctccaa ggcgtacatc aagcaccccg ccgacatccc
     4501 cgactacctc aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     4561 ggacggcggc gtggtgaccg tgacccagga ctcatccctg caagacggca ccctcatcta
     4621 ccacgtcaag ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc agaagaaaac
     4681 tcttggctgg gagccctcca ctgagcgcaa ctacccccgc gacggcgtgc tgaagggcga
     4741 gaaccacatg gcgctgaagc tgaagggcgg cggccactac ctgtgtgagt tcaagtccat
     4801 ctacatggct aagaagcccg tgaagctgcc cggctaccac tacgtggact acaagctcga
     4861 catcacctcc cacaacgagg actacaccgt ggtggagcag tacgaacgcg ccgaggcccg
     4921 ccaccacctg ttccagtagg tttaaacatc aacctctgga ttacaaaatt tgtgaaagat
     4981 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     5041 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     5101 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     5161 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     5221 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     5281 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     5341 agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     5401 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     5461 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     5521 gggccgcctc cccgcctggc ctgcaggggt acctttaaga ccaatgactt acaaggcagc
     5581 tgtagatctt agccactttt taaaagaaaa gaggggactg gaagggctaa ttcactccca
     5641 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgatta
     5701 gcagaactac acaccagggc caggggtcag atatccactg acctttggat ggtgctacaa
     5761 gctagtacca gttgagccag ataaggtaga agaggccaat aaaggagaga acaccagctt
     5821 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtgt tagagtggag
     5881 gtttgacagc cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg agtacttcaa
     5941 gaactgctga tatcgagctt gctacaaggg actttccgct ggggactttc cagggaggcg
     6001 tggcctgggc gggactgggg agtggcgagc cctcagatcc tgcatataag cagctgcttt
     6061 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     6121 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     6181 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     6241 aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     6301 aatgaatatc agagagtgag aggccttgac attgctagcg ttttaccgtc gacctctagc
     6361 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6421 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6481 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6541 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6601 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7201 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7381 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7441 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7501 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7561 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7621 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7681 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7741 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7801 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7861 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7921 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7981 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     8041 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     8101 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     8161 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     8221 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     8281 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8341 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8401 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8461 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8521 aaagtgccac ctgacgtcga cggatcggga gatcaacttg tttattgcag cttataatgg
     8581 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     8641 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca actggataac
     8701 tcaagctaac caaaatcatc ccaaacttcc caccccatac cctattacca ctgccaatta
     8761 cctgtggttt catttactct aaacctgtga ttcctctgaa ttattttcat tttaaagaaa
     8821 ttgtatttgt taaatatgta ctacaaactt agtagt
//
