LOCUS       sequence-430238-        9574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with mOrange reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9574)
  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 9574)
  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: LEAPOW
FEATURES             Location/Qualifiers
     source          1..9574
                     /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"
     intron          2397..3339
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3296..3316
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        4430..4929
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4465..4488)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4862..4881
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(5718..5738)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6136..6147)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6337..6970
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(7099..7115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7099..7115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7112..7134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7123..7139
                     /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(7147..7153,7154..7171,7172..7177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7192..7213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7330..7347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7501..8089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7581..7600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8260..9051,9052..9120))
                     /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     8883..8902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9121..9225)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    9273..9407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9310..9329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9364..9383
                     /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 acacaggtaa
     2401 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     2461 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2521 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2581 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2641 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2701 tttttctggc aagatagtct tgtaaatgcg ggccaagatc ggcacactgg tatttcggtt
     2761 tttggggccg cgggcggcga cggggccggt gcgtcccagc gcacatgttc ggcgaggcgg
     2821 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2881 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2941 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3001 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3061 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3121 cacctcgatt agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3181 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3241 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3301 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaag cgcttcgact
     3361 agggataaca gggtaattgt ttgaatgagg cttcagtact ttacagaatc gttgcctgca
     3421 catcttggaa acacttgctg ggattacttc gacttcttaa cccaacagaa ggctcgagaa
     3481 ggtatattgc tgttgacagg atccacatta acaactttaa gtccagcaat ttgagttaag
     3541 ggtgttgctc tcaatgattt cactaatggt tcaattttta atttcttttc ttctggttca
     3601 aaattcaagt ttcaagtgaa agtgtaatat gcacccattt ctttcaataa atcttctaaa
     3661 tagtccacta atgttttatt ttgtttttta caaaatcaag cagcctctgc tatcaatata
     3721 gaaccttgta ttccatcttt atctctagct gagtcatcaa ttacatatcc ataactttct
     3781 tcataagcaa aaacaaaatt taatccgtta tcttcttctt tagcaatttc tctacccatt
     3841 catttcaatc cagttaaagt ttttacaatc ttaactccat atttttcctg agcgattcta
     3901 tcacccaaat cacttgttac aaaacttgaa tatagagccg gattttttgg aatgctattt
     3961 aagcgtttta gatttgataa ttttcaatca attaaaattg gtcctgtttg atttccatct
     4021 aatcttacaa aatgaccatc atgttttatt gccattccaa atctgtcagc atctgggtca
     4081 ttcataataa taatatctgc atcatgttta ataccatatt caagcggtat ttttcatgca
     4141 ggatcaaatt ctggatttgg atttacaaca tttttcaatg tttcatcttc aaatgcacga
     4201 tcttcaccct caataacgtt atatcctgat tcacgtaatc tttttggggt aaatttagtt
     4261 cctgttccat taactgcgct aaaaataggg ccctgcctcg gacttcaagg ggctagaatt
     4321 cgagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat acatttttac
     4381 aaagctgaat taaaatggta taaattaaat cacttttttc aattctaccg ggtaggggag
     4441 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     4501 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     4561 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     4621 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     4681 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     4741 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     4801 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     4861 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     4921 ggcctttcga cctttaatta accaccatgg tgagcaaggg cgaagagaat aacatggcaa
     4981 tcatcaagga gttcatgcgc ttcaaggttc gcatggaggg ctccgtgaac ggccacgagt
     5041 tcgagatcga gggcgagggc gagggccgcc cctacgaggg ctttcagacc gctaagctga
     5101 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcacct
     5161 acggctccaa ggcatacgtg aagcaccccg ccgacatccc cgactacttc aagctgtcct
     5221 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     5281 tgacccagga ctcgtccctg caagacggcg agttcatcta caaggtgaag ctgcgcggca
     5341 ccaacttccc ctccgacggc cccgtaatgc agaaaaagac tatgggctgg gaggcatcct
     5401 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     5461 tgaaggacgg cggccactac acctccgagg tcaagaccac ctacaaggcc aagaagcccg
     5521 tgcaactgcc cggcgcctac atcgtcggca tcaagttgga catcacctcc cacaacgagg
     5581 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     5641 acgagctata caagtaggtt taaacatcaa cctctggatt acaaaatttg tgaaagattg
     5701 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5761 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5821 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5881 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5941 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     6001 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     6061 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     6121 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     6181 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     6241 gccgcctccc cgcctggcct gcaggggtac ctttaagacc aatgacttac aaggcagctg
     6301 tagatcttag ccacttttta aaagaaaaga ggggactgga agggctaatt cactcccaac
     6361 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc
     6421 agaactacac accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc
     6481 tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt
     6541 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt
     6601 ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga
     6661 actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg
     6721 gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt
     6781 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     6841 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     6901 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     6961 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     7021 tgaatatcag agagtgagag gccttgacat tgctagcgtt ttaccgtcga cctctagcta
     7081 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     7141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     7201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     7321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7921 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     8041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     8101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     8161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     8221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     8281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     8341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     8641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     8701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     9001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     9061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     9121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     9181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     9241 agtgccacct gacgtcgacg gatcgggaga tcaacttgtt tattgcagct tataatggtt
     9301 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     9361 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc
     9421 aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc
     9481 tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt
     9541 gtatttgtta aatatgtact acaaacttag tagt
//
