LOCUS       sequence-430320-        9215 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 9215)
  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 9215)
  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: LBAPTW
FEATURES             Location/Qualifiers
     source          1..9215
                     /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        2187..2472
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2474..2751
                     /label=chicken beta-actin promoter
     promoter        4067..4566
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4102..4125)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4499..4518
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4584..5294
                     /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(5358..5378)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5776..5787)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5977..6610
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6739..6755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6739..6755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6752..6774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6763..6779
                     /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(6787..6793,6794..6811,6812..6817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6832..6853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6970..6987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7141..7729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7221..7240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7900..8691,8692..8760))
                     /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     8523..8542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8761..8865)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8913..9047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8950..8969)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9004..9023
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(9215,1..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 gatagtcgtt acataactta cggtaaatgg cccgcctggc
     2221 tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat
     2281 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2341 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt
     2401 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2461 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2521 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2581 gggggggggg gggggcgcgc gccrgggggg gsggggsggg gsgrggggsg gggsggggsg
     2641 rggcggagag gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2701 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2761 gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     2821 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     2881 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     2941 taattacctg ttttacaggc ctgaaatcac ttggttttag gttggagcgc ttcgactagg
     3001 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     3061 cttggaaaca cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt
     3121 atattgctgt tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt
     3181 gttgctctca atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa
     3241 ttcaagtttc aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag
     3301 tccactaatg ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa
     3361 ccttgtattc catctttatc tctagctgag tcatcaatta catatccata actttcttca
     3421 taagcaaaaa caaaatttaa tccgttatct tcttctttag caatttctct acccattcat
     3481 ttcaatccag ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca
     3541 cccaaatcac ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag
     3601 cgttttagat ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat
     3661 cttacaaaat gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc
     3721 ataataataa tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga
     3781 tcaaattctg gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct
     3841 tcaccctcaa taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct
     3901 gttccattaa ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga
     3961 gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa
     4021 gctgaattaa aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg
     4081 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     4141 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
     4201 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     4261 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
     4321 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     4381 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     4441 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     4501 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     4561 ctttcgacct ttaattaacc accatggtgt ctaagggcga agagctgatt aaggagaaca
     4621 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     4681 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     4741 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     4801 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     4861 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     4921 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     4981 acggccctgt gatgcagaag aaaacactcg gctgggaggc gttcaccgag acgctgtacc
     5041 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     5101 atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga
     5161 tgcctggcgt gtactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     5221 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     5281 ggcacaagct aaattaggtt taaacatcaa cctctggatt acaaaatttg tgaaagattg
     5341 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5401 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5461 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5521 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5581 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     5641 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     5701 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     5761 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     5821 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     5881 gccgcctccc cgcctggcct gcaggggtac ctttaagacc aatgacttac aaggcagctg
     5941 tagatcttag ccacttttta aaagaaaaga ggggactgga agggctaatt cactcccaac
     6001 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc
     6061 agaactacac accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc
     6121 tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt
     6181 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt
     6241 ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga
     6301 actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg
     6361 gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt
     6421 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     6481 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     6541 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     6601 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     6661 tgaatatcag agagtgagag gccttgacat tgctagcgtt ttaccgtcga cctctagcta
     6721 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6781 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6841 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6961 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7021 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7741 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7801 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7981 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     8281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     8341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8461 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8761 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8821 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8881 agtgccacct gacgtcgacg gatcgggaga tcaacttgtt tattgcagct tataatggtt
     8941 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     9001 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc
     9061 aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc
     9121 tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt
     9181 gtatttgtta aatatgtact acaaacttag tagtt
//
