LOCUS       sequence-430326-        9024 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 9024)
  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 9024)
  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: LCAPTW
FEATURES             Location/Qualifiers
     source          1..9024
                     /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="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(5168..5188)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5586..5597)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5787..6420
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6549..6565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6549..6565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6562..6584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6573..6589
                     /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(6597..6603,6604..6621,6622..6627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6642..6663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6780..6797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6951..7539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7031..7050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7710..8501,8502..8570))
                     /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     8333..8352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8571..8675)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8723..8857
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8760..8779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8814..8833
                     /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 accatggtgt ctaagggcga agagctgatt aaggagaaca tgcacatgaa
     4441 gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg
     4501 caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc
     4561 cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca
     4621 cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca catgggagag
     4681 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     4741 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt
     4801 gatgcagaag aaaacactcg gctgggaggc gttcaccgag acgctgtacc ccgctgacgg
     4861 cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc
     4921 aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt
     4981 gtactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga
     5041 gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct
     5101 aaattaggtt taaacatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5161 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5221 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     5281 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     5341 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     5401 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     5461 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     5521 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     5581 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     5641 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     5701 cgcctggcct gcaggggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     5761 ccacttttta aaagaaaaga ggggactgga agggctaatt cactcccaac gaagacaaga
     5821 tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc agaactacac
     5881 accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     5941 tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt
     6001 gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg
     6061 cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata
     6121 tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg
     6181 gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg
     6241 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     6301 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     6361 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     6421 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     6481 agagtgagag gccttgacat tgctagcgtt ttaccgtcga cctctagcta gagcttggcg
     6541 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6601 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6661 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6721 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6781 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6841 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6901 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6961 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     7021 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     7081 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     7141 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     7201 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     7261 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     7321 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7381 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7441 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7501 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7561 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7621 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7681 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7741 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7801 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7861 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7921 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7981 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     8041 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     8101 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     8161 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     8221 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     8281 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     8341 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     8401 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8461 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8521 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8581 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8641 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8701 gacgtcgacg gatcgggaga tcaacttgtt tattgcagct tataatggtt acaaataaag
     8761 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     8821 gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca
     8881 aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca
     8941 tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta
     9001 aatatgtact acaaacttag tagt
//
