LOCUS       sequence-409133-        7997 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 7997)
  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 7997)
  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: MAPCW
FEATURES             Location/Qualifiers
     source          1..7997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2494..2993
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2529..2552)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2926..2945
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3011..3721
                     /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(3156..3174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3782..3802)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4200..4211)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4392..4906
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5068..5084
                     /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(5092..5098,5099..5116,5117..5122))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5137..5158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5275..5292)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5446..6034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5526..5545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6205..6996,6997..7065))
                     /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     6828..6847
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7066..7170)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7238..7256
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7294..7316)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7416..7435
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7629..7651
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7779..7798
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatctctc gactagggat aacagggtaa
     1441 ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt
     1501 gctgggatta cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga
     1561 caggatccac attaacaact ttaagtccag caatttgagt taagggtgtt gctctcaatg
     1621 atttcactaa tggttcaatt tttaatttct tttcttctgg ttcaaaattc aagtttcaag
     1681 tgaaagtgta atatgcaccc atttctttca ataaatcttc taaatagtcc actaatgttt
     1741 tattttgttt tttacaaaat caagcagcct ctgctatcaa tatagaacct tgtattccat
     1801 ctttatctct agctgagtca tcaattacat atccataact ttcttcataa gcaaaaacaa
     1861 aatttaatcc gttatcttct tctttagcaa tttctctacc cattcatttc aatccagtta
     1921 aagtttttac aatcttaact ccatattttt cctgagcgat tctatcaccc aaatcacttg
     1981 ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt tttagatttg
     2041 ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt acaaaatgac
     2101 catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata ataataatat
     2161 ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca aattctggat
     2221 ttggatttac aacatttttc aatgtttcat cttcaaatgc acgatcttca ccctcaataa
     2281 cgttatatcc tgattcacgt aatctttttg gggtaaattt agttcctgtt ccattaactg
     2341 cgctaaaaat agggccctgc ctcggacttc aaggggctag aattcgagca attatcttgt
     2401 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     2461 ggtataaatt aaatcacttt tttcaattct accgggtagg ggaggcgctt ttcccaaggc
     2521 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     2581 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     2641 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     2701 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     2761 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     2821 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     2881 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     2941 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgaccttta
     3001 attaaccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     3061 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     3121 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     3181 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggcata
     3241 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     3301 ctgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     3361 cctacaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3421 cggccccgta atgcagtgtc gtacaatggg ctgggaggct tccactgagc ggatgtaccc
     3481 cgaggacggt gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     3541 ctacgacgct gaagtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggagc
     3601 ctacaacgtg gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga
     3661 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     3721 ggtttaaaca tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3781 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3841 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3901 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3961 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     4021 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     4081 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     4141 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     4201 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     4261 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     4321 ggtcgacctg caggcatgca agcttatcga taaaataaaa gattttattt agtctccaga
     4381 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
     4441 tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac
     4501 agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     4561 tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac
     4621 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
     4681 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
     4741 agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc
     4801 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
     4861 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct
     4921 tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat
     4981 tagccactgt tttgaatcca catactccaa tactcctgaa atagttcatt atggacagcg
     5041 cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac acaacatacg
     5101 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5161 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5221 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5281 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5341 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5401 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5461 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5521 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5581 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5641 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5701 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5761 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5821 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5881 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5941 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6001 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6061 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6121 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6181 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6241 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6301 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6361 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6421 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6481 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6541 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6601 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6661 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6721 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6781 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6841 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6901 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6961 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7021 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7081 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7141 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7201 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7261 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     7321 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     7381 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     7441 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     7501 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     7561 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     7621 agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc atgcaaggag
     7681 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7741 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     7801 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggcga
     7861 ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa
     7921 tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt
     7981 ctccagaaaa agggggg
//
