LOCUS       sequence-409141-        7964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with E2-Crimson reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7964)
  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 7964)
  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: MAPEW
FEATURES             Location/Qualifiers
     source          1..7964
                     /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..3688
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3749..3769)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4167..4178)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4359..4873
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5035..5051
                     /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(5059..5065,5066..5083,5084..5089))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5104..5125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5242..5259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5413..6001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5493..5512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6172..6963,6964..7032))
                     /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     6795..6814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7033..7137)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7205..7223
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7261..7283)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7383..7402
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7596..7618
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7746..7765
                     /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 atggatagca ctgagaacgt catcaagccc ttcatgcgct tcaaggtgca
     3061 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgtgggcg agggcaagcc
     3121 ctacgagggc acccagaccg ccaagctgca agtgaccaag ggcggccccc tgcccttcgc
     3181 ctgggacatc ctgtcccccc agttcttcta cggctccaag gcgtacatca agcaccccgc
     3241 cgacatcccc gactacctca agcagtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3301 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcatccctgc aagacggcac
     3361 cctcatctac cacgtcaagt tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca
     3421 gaagaaaact cttggctggg agccctccac tgagcgcaac tacccccgcg acggcgtgct
     3481 gaagggcgag aaccacatgg cgctgaagct gaagggcggc ggccactacc tgtgtgagtt
     3541 caagtccatc tacatggcta agaagcccgt gaagctgccc ggctaccact acgtggacta
     3601 caagctcgac atcacctccc acaacgagga ctacaccgtg gtggagcagt acgaacgcgc
     3661 cgaggcccgc caccacctgt tccagtaggt ttaaacatca acctctggat tacaaaattt
     3721 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3781 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3841 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3901 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3961 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4021 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4081 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
     4141 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4201 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4261 tctccctttg ggccgcctcc ccgcctgggt cgacctgcag gcatgcaagc ttatcgataa
     4321 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     4381 ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat
     4441 agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata
     4501 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     4561 tcccgccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     4621 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     4681 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct
     4741 ccgatagact gcgtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     4801 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     4861 gggggtcttt catgggtaac agtttcttga agttggagaa caacattctg agggtaggag
     4921 tcgaatatta agtaatcctg actcaattag ccactgtttt gaatccacat actccaatac
     4981 tcctgaaata gttcattatg gacagcgcag aaagagctgg ggagaattgt gaaattgtta
     5041 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5101 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5161 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5221 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5281 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5341 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5401 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5461 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5521 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5581 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5641 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5701 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5761 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5821 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5881 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5941 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6001 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6061 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6121 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6181 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6241 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6301 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6361 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6421 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6481 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6541 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6601 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6661 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6721 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6781 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6841 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6901 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6961 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7021 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7081 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7141 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     7201 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     7261 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     7321 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     7381 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     7441 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     7501 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     7561 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     7621 ggcgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     7681 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     7741 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     7801 gccacgatgc gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg
     7861 gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca
     7921 tagataaaat aaaagatttt atttagtctc cagaaaaagg gggg
//
