LOCUS       sequence-409390-        9212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with mCherry reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9212)
  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 9212)
  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: LBAPCW
FEATURES             Location/Qualifiers
     source          1..9212
                     /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        2188..2473
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2475..2752
                     /label=chicken beta-actin promoter
     promoter        4068..4567
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4103..4126)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4500..4519
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4585..5295
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4730..4748)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5356..5376)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5774..5785)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5975..6608
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6737..6753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6737..6753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6750..6772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6761..6777
                     /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(6785..6791,6792..6809,6810..6815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6830..6851
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6968..6985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7139..7727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7219..7238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7898..8689,8690..8758))
                     /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     8521..8540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8759..8863)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8911..9045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8948..8967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9002..9021
                     /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 cgatagtcgt tacataactt acggtaaatg gcccgcctgg
     2221 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
     2281 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2341 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
     2401 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2461 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     2521 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     2581 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
     2641 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     2701 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     2761 tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     2821 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     2881 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     2941 ttaattacct gttttacagg cctgaaatca cttggtttta ggttggagcg cttcgactag
     3001 ggataacagg gtaattgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca
     3061 tcttggaaac acttgctggg attacttcga cttcttaacc caacagaagg ctcgagaagg
     3121 tatattgctg ttgacaggat ccacattaac aactttaagt ccagcaattt gagttaaggg
     3181 tgttgctctc aatgatttca ctaatggttc aatttttaat ttcttttctt ctggttcaaa
     3241 attcaagttt caagtgaaag tgtaatatgc acccatttct ttcaataaat cttctaaata
     3301 gtccactaat gttttatttt gttttttaca aaatcaagca gcctctgcta tcaatataga
     3361 accttgtatt ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc
     3421 ataagcaaaa acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca
     3481 tttcaatcca gttaaagttt ttacaatctt aactccatat ttttcctgag cgattctatc
     3541 acccaaatca cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa
     3601 gcgttttaga tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa
     3661 tcttacaaaa tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt
     3721 cataataata atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg
     3781 atcaaattct ggatttggat ttacaacatt tttcaatgtt tcatcttcaa atgcacgatc
     3841 ttcaccctca ataacgttat atcctgattc acgtaatctt tttggggtaa atttagttcc
     3901 tgttccatta actgcgctaa aaatagggcc ctgcctcgga cttcaagggg ctagaattcg
     3961 agcaattatc ttgtttacta aaactgaata ccttgctatc tctttgatac atttttacaa
     4021 agctgaatta aaatggtata aattaaatca cttttttcaa ttctaccggg taggggaggc
     4081 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     4141 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     4201 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     4261 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     4321 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     4381 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
     4441 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
     4501 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
     4561 cctttcgacc tttaattaac caccatggtg agcaagggcg aggaggataa catggccatc
     4621 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     4681 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     4741 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     4801 ggctccaagg catacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     4861 cccgagggct tcaactggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     4921 acccaggact cctccctaca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     4981 aacttcccct ccgacggccc cgtaatgcag tgtcgtacaa tgggctggga ggcttccact
     5041 gagcggatgt accccgagga cggtgccctg aagggcgaga tcaagcagag gctgaagctg
     5101 aaggacggcg gccactacga cgctgaagtc aagaccacct acaaggccaa gaagcccgtg
     5161 cagctgcccg gagcctacaa cgtggacatc aagttggaca tcctttccca caacgaggac
     5221 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     5281 gagctgtaca agtaggttta aacatcaacc tctggattac aaaatttgtg aaagattgac
     5341 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     5401 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     5461 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     5521 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     5581 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     5641 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     5701 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     5761 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     5821 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     5881 cgcctccccg cctggcctgc aggggtacct ttaagaccaa tgacttacaa ggcagctgta
     5941 gatcttagcc actttttaaa agaaaagagg ggactggaag ggctaattca ctcccaacga
     6001 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattagcag
     6061 aactacacac cagggccagg ggtcagatat ccactgacct ttggatggtg ctacaagcta
     6121 gtaccagttg agccagataa ggtagaagag gccaataaag gagagaacac cagcttgtta
     6181 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtgttaga gtggaggttt
     6241 gacagccgcc tagcatttca tcacgtggcc cgagagctgc atccggagta cttcaagaac
     6301 tgctgatatc gagcttgcta caagggactt tccgctgggg actttccagg gaggcgtggc
     6361 ctgggcggga ctggggagtg gcgagccctc agatcctgca tataagcagc tgctttttgc
     6421 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     6481 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     6541 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     6601 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     6661 aatatcagag agtgagaggc cttgacattg ctagcgtttt accgtcgacc tctagctaga
     6721 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     6781 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     6841 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6901 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6961 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     7021 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     7081 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     7141 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     7201 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     7261 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     7321 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     7381 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     7441 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     7501 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     7561 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     7621 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     7681 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     7741 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7801 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7861 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7921 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7981 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     8041 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     8101 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     8161 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     8221 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     8281 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     8341 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     8401 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     8461 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     8521 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     8581 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     8641 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     8701 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     8761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     8821 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     8881 tgccacctga cgtcgacgga tcgggagatc aacttgttta ttgcagctta taatggttac
     8941 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     9001 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaactg gataactcaa
     9061 gctaaccaaa atcatcccaa acttcccacc ccatacccta ttaccactgc caattacctg
     9121 tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta aagaaattgt
     9181 atttgttaaa tatgtactac aaacttagta gt
//
