LOCUS       sequence-430239-        8892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with mTagBFP2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8892)
  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 8892)
  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: LHAPTW
FEATURES             Location/Qualifiers
     source          1..8892
                     /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..4972
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(5036..5056)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5454..5465)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5655..6288
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6417..6433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6417..6433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6430..6452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6441..6457
                     /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(6465..6471,6472..6489,6490..6495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6510..6531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6648..6665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6819..7407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6899..6918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7578..8369,8370..8438))
                     /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     8201..8220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8439..8543)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8591..8725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8628..8647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8682..8701
                     /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 catggtgtct aagggcgaag agctgattaa ggagaacatg cacatgaagc tgtacatgga
     4321 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     4381 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     4441 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     4501 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     4561 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     4621 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     4681 aacactcggc tgggaggcgt tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     4741 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acgccaagac
     4801 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtgt actatgtgga
     4861 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     4921 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctaa attaggttta
     4981 aacatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     5041 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     5101 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     5161 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     5221 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     5281 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     5341 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     5401 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     5461 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     5521 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggcctgc
     5581 aggggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5641 agaaaagagg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5701 gtggatctac cacacacaag gctacttccc tgattagcag aactacacac cagggccagg
     5761 ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa
     5821 ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg
     5881 gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca
     5941 tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta
     6001 caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg
     6061 gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt
     6121 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     6181 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6241 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     6301 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc
     6361 cttgacattg ctagcgtttt accgtcgacc tctagctaga gcttggcgta atcatggtca
     6421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     8581 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     8641 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     8701 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     8761 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     8821 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     8881 aaacttagta gt
//
