LOCUS       sequence-409382-        8910 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 8910)
  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 8910)
  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: LCAPPW
FEATURES             Location/Qualifiers
     source          1..8910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    680..805
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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    2027..2142
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     enhancer        2200..2503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2504..2707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2658..2678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2704..2728
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3876..4375
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3911..3934)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4308..4327
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4393..4992
                     /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(4393..4412)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4889..4909
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5053..5073)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5471..5482)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5672..6305
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6434..6450)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6434..6450)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6447..6469)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6458..6474
                     /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(6482..6488,6489..6506,6507..6512))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6527..6548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6665..6682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6836..7424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6916..6935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7595..8386,8387..8455))
                     /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     8218..8237
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8456..8560)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8608..8742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8645..8664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8699..8718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(8910,1..633)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac
       61 acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact
      121 gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa
      181 taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga
      241 gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga
      301 gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc
      361 tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc
      421 ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag
      481 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      541 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      601 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      661 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      721 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      781 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      841 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      901 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      961 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1021 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1081 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1141 agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1981 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccttta aaagaaaagg
     2041 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2101 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2161 cagcagagat ccagtttatc gataagcttg ggagttccgc gttacataac ttacggtaaa
     2221 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     2281 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2341 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2401 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2461 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2521 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2581 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2641 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2701 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
     2761 ccatagaaga caccgactct actagaggat cgctagcgct tcgactaggg ataacagggt
     2821 aattgtttga atgaggcttc agtactttac agaatcgttg cctgcacatc ttggaaacac
     2881 ttgctgggat tacttcgact tcttaaccca acagaaggct cgagaaggta tattgctgtt
     2941 gacaggatcc acattaacaa ctttaagtcc agcaatttga gttaagggtg ttgctctcaa
     3001 tgatttcact aatggttcaa tttttaattt cttttcttct ggttcaaaat tcaagtttca
     3061 agtgaaagtg taatatgcac ccatttcttt caataaatct tctaaatagt ccactaatgt
     3121 tttattttgt tttttacaaa atcaagcagc ctctgctatc aatatagaac cttgtattcc
     3181 atctttatct ctagctgagt catcaattac atatccataa ctttcttcat aagcaaaaac
     3241 aaaatttaat ccgttatctt cttctttagc aatttctcta cccattcatt tcaatccagt
     3301 taaagttttt acaatcttaa ctccatattt ttcctgagcg attctatcac ccaaatcact
     3361 tgttacaaaa cttgaatata gagccggatt ttttggaatg ctatttaagc gttttagatt
     3421 tgataatttt caatcaatta aaattggtcc tgtttgattt ccatctaatc ttacaaaatg
     3481 accatcatgt tttattgcca ttccaaatct gtcagcatct gggtcattca taataataat
     3541 atctgcatca tgtttaatac catattcaag cggtattttt catgcaggat caaattctgg
     3601 atttggattt acaacatttt tcaatgtttc atcttcaaat gcacgatctt caccctcaat
     3661 aacgttatat cctgattcac gtaatctttt tggggtaaat ttagttcctg ttccattaac
     3721 tgcgctaaaa atagggccct gcctcggact tcaaggggct agaattcgag caattatctt
     3781 gtttactaaa actgaatacc ttgctatctc tttgatacat ttttacaaag ctgaattaaa
     3841 atggtataaa ttaaatcact tttttcaatt ctaccgggta ggggaggcgc ttttcccaag
     3901 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     3961 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     4021 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     4081 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     4141 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     4201 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     4261 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     4321 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctt
     4381 taattaacca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     4441 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     4501 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     4561 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     4621 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     4681 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     4741 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     4801 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     4861 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     4921 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     4981 cccggtgcct gagtttaaac atcaacctct ggattacaaa atttgtgaaa gattgactgg
     5041 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     5101 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     5161 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     5221 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     5281 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     5341 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     5401 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     5461 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     5521 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     5581 ctccccgcct ggcctgcagg ggtaccttta agaccaatga cttacaaggc agctgtagat
     5641 cttagccact ttttaaaaga aaagagggga ctggaagggc taattcactc ccaacgaaga
     5701 caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac
     5761 tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta
     5821 ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac
     5881 cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac
     5941 agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc
     6001 tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg
     6061 ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg
     6121 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     6181 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     6241 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     6301 tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     6361 atcagagagt gagaggcctt gacattgcta gcgttttacc gtcgacctct agctagagct
     6421 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6481 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6541 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6601 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6661 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6721 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6781 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6841 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6901 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6961 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7021 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7081 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7141 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7201 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7261 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7321 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7381 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7441 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7501 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7561 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7621 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7681 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7741 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7801 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7861 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7921 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7981 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8041 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8101 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8161 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8221 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8281 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8341 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8401 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8461 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8521 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8581 cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa tggttacaaa
     8641 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     8701 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat aactcaagct
     8761 aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa ttacctgtgg
     8821 tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag aaattgtatt
     8881 tgttaaatat gtactacaaa cttagtagtt
//
