LOCUS       sequence-409379-        7886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with Puromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7886)
  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 7886)
  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: MAPPW
FEATURES             Location/Qualifiers
     source          1..7886
                     /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..3610
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3011..3030)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3507..3527
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3671..3691)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4089..4100)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4281..4795
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4957..4973
                     /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(4981..4987,4988..5005,5006..5011))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5026..5047
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5164..5181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5335..5923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5415..5434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6094..6885,6886..6954))
                     /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     6717..6736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6955..7059)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7127..7145
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7183..7205)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7305..7324
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7518..7540
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7668..7687
                     /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 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     3061 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     3121 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     3181 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     3241 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     3301 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     3361 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     3421 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     3481 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     3541 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     3601 cggtgcctga gtttaaacat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3661 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3721 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3781 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3841 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3901 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3961 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     4021 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     4081 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4141 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4201 ccccgcctgg gtcgacctgc aggcatgcaa gcttatcgat aaaataaaag attttattta
     4261 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     4321 taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag
     4381 gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc
     4441 tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt
     4501 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     4561 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     4621 caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc
     4681 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     4741 tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatgggta
     4801 acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc
     4861 tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa tagttcatta
     4921 tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca caattccaca
     4981 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5041 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5101 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5161 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5221 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5281 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5341 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5401 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5461 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5521 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5581 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5641 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5701 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5761 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5821 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5881 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5941 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6001 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6061 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6121 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6181 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6241 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6301 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6361 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6421 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6481 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6541 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6601 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6661 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6721 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6781 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6841 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6901 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6961 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7021 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7081 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     7141 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     7201 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     7261 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     7321 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     7381 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     7441 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     7501 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca
     7561 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     7621 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     7681 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     7741 tagaggcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg
     7801 actcaacaat atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt
     7861 ttatttagtc tccagaaaaa gggggg
//
