LOCUS       sequence-409383-        8955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with Venus reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8955)
  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 8955)
  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: LMAPVW
FEATURES             Location/Qualifiers
     source          1..8955
                     /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..2719
                     /label=mCMV promoter
                     /note="mouse cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        3801..4300
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3836..3859)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4233..4252
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(4318..4320,4321..4323,4324..5037)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4363..4384)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4624..4643)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4971..4992
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5098..5118)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5516..5527)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5717..6350
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6479..6495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6479..6495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6492..6514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6503..6519
                     /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(6527..6533,6534..6551,6552..6557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6572..6593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6710..6727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6881..7469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6961..6980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7640..8431,8432..8500))
                     /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     8263..8282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8501..8605)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8653..8787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8690..8709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8744..8763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(8955,1..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 gattggcacg tatactgagt cattagggac tttccaatgg
     2221 gttttgccca gtacataagg tcaatagggg tgaatcaaca ggaaagtccc attggagcca
     2281 agtacactga gtcaataggg actttccatt gggttttgcc cagtacaaaa ggtcaatagg
     2341 gggtgagtca atgggttttt cccattattg gcacgtacat aaggtcaata ggggtgagtc
     2401 attgggtttt tccagccaat ttaattataa cgccatgtac tttcccacca ttgacgtcaa
     2461 tgggctattg aaactaatgc aacgtgacct ttaaacggta ctttcccata gctgattaat
     2521 gggaaagtac cgttgtcgag ccaatacacg tcaatgggaa gtgaaagggc agccaaaacg
     2581 taacaccgcc ccggttttcc cctggaaatt ccatattggc acgcattcta ttggctgagc
     2641 tgcgttctac gtgggtataa gaggcgcgac cagcgtccgt accgtcgcag tcttcggtct
     2701 gaccaccgta gaacgcagaa gcgcttcgac tagggataac agggtaattg tttgaatgag
     2761 gcttcagtac tttacagaat cgttgcctgc acatcttgga aacacttgct gggattactt
     2821 cgacttctta acccaacaga aggctcgaga aggtatattg ctgttgacag gatccacatt
     2881 aacaacttta agtccagcaa tttgagttaa gggtgttgct ctcaatgatt tcactaatgg
     2941 ttcaattttt aatttctttt cttctggttc aaaattcaag tttcaagtga aagtgtaata
     3001 tgcacccatt tctttcaata aatcttctaa atagtccact aatgttttat tttgtttttt
     3061 acaaaatcaa gcagcctctg ctatcaatat agaaccttgt attccatctt tatctctagc
     3121 tgagtcatca attacatatc cataactttc ttcataagca aaaacaaaat ttaatccgtt
     3181 atcttcttct ttagcaattt ctctacccat tcatttcaat ccagttaaag tttttacaat
     3241 cttaactcca tatttttcct gagcgattct atcacccaaa tcacttgtta caaaacttga
     3301 atatagagcc ggattttttg gaatgctatt taagcgtttt agatttgata attttcaatc
     3361 aattaaaatt ggtcctgttt gatttccatc taatcttaca aaatgaccat catgttttat
     3421 tgccattcca aatctgtcag catctgggtc attcataata ataatatctg catcatgttt
     3481 aataccatat tcaagcggta tttttcatgc aggatcaaat tctggatttg gatttacaac
     3541 atttttcaat gtttcatctt caaatgcacg atcttcaccc tcaataacgt tatatcctga
     3601 ttcacgtaat ctttttgggg taaatttagt tcctgttcca ttaactgcgc taaaaatagg
     3661 gccctgcctc ggacttcaag gggctagaat tcgagcaatt atcttgttta ctaaaactga
     3721 ataccttgct atctctttga tacattttta caaagctgaa ttaaaatggt ataaattaaa
     3781 tcactttttt caattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     3841 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     3901 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     3961 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     4021 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     4081 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     4141 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     4201 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     4261 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctttaatt aaccaccatg
     4321 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     4381 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     4441 aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     4501 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     4561 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     4621 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     4681 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     4741 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     4801 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     4861 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     4921 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     4981 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagtt
     5041 taaacatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5101 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     5161 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     5221 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     5281 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     5341 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     5401 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     5461 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     5521 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     5581 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggcct
     5641 gcaggggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     5701 aaagaaaaga ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
     5761 ctgtggatct accacacaca aggctacttc cctgattagc agaactacac accagggcca
     5821 ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat
     5881 aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat
     5941 gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt
     6001 catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc
     6061 tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag
     6121 tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg
     6181 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     6241 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     6301 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     6361 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     6421 gccttgacat tgctagcgtt ttaccgtcga cctctagcta gagcttggcg taatcatggt
     6481 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     6541 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6601 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6661 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6721 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6781 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6841 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6901 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6961 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     7021 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     7081 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     7141 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     7201 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     7261 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     7321 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     7381 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7441 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7501 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7561 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7621 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7681 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7741 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7801 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7861 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7921 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7981 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     8041 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     8101 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     8161 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     8221 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     8281 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     8341 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     8401 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8461 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8521 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8581 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     8641 gatcgggaga tcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     8701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     8761 atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc
     8821 aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa
     8881 acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact
     8941 acaaacttag tagtt
//
