LOCUS       sequence-409393-        9101 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 9101)
  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 9101)
  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: LBAPPW
FEATURES             Location/Qualifiers
     source          1..9101
                     /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        2187..2472
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2474..2751
                     /label=chicken beta-actin promoter
     promoter        4067..4566
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4102..4125)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4499..4518
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4584..5183
                     /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(4584..4603)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5080..5100
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5244..5264)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5662..5673)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5863..6496
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6625..6641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6625..6641)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6638..6660)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6649..6665
                     /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(6673..6679,6680..6697,6698..6703))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6718..6739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6856..6873)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7027..7615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7107..7126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7786..8577,8578..8646))
                     /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     8409..8428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8647..8751)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8799..8933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8836..8855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8890..8909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(9101,1..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 gatagtcgtt acataactta cggtaaatgg cccgcctggc
     2221 tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat
     2281 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2341 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt
     2401 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2461 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2521 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2581 gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
     2641 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2701 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2761 gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     2821 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     2881 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     2941 taattacctg ttttacaggc ctgaaatcac ttggttttag gttggagcgc ttcgactagg
     3001 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     3061 cttggaaaca cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt
     3121 atattgctgt tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt
     3181 gttgctctca atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa
     3241 ttcaagtttc aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag
     3301 tccactaatg ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa
     3361 ccttgtattc catctttatc tctagctgag tcatcaatta catatccata actttcttca
     3421 taagcaaaaa caaaatttaa tccgttatct tcttctttag caatttctct acccattcat
     3481 ttcaatccag ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca
     3541 cccaaatcac ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag
     3601 cgttttagat ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat
     3661 cttacaaaat gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc
     3721 ataataataa tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga
     3781 tcaaattctg gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct
     3841 tcaccctcaa taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct
     3901 gttccattaa ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga
     3961 gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa
     4021 gctgaattaa aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg
     4081 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     4141 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
     4201 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     4261 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
     4321 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     4381 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     4441 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     4501 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     4561 ctttcgacct ttaattaacc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4621 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4681 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4741 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4801 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     4861 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     4921 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     4981 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     5041 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     5101 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     5161 tgacccgcaa gcccggtgcc tgagtttaaa catcaacctc tggattacaa aatttgtgaa
     5221 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     5281 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     5341 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     5401 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     5461 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     5521 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     5581 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg
     5641 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     5701 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     5761 ctttgggccg cctccccgcc tggcctgcag gggtaccttt aagaccaatg acttacaagg
     5821 cagctgtaga tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact
     5881 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     5941 attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct
     6001 acaagctagt accagttgag ccagataagg tagaagaggc caataaagga gagaacacca
     6061 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt
     6121 ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact
     6181 tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga
     6241 ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg
     6301 ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     6361 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     6421 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     6481 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     6541 aagaaatgaa tatcagagag tgagaggcct tgacattgct agcgttttac cgtcgacctc
     6601 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6661 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6721 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6781 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6841 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6901 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6961 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     7021 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7081 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7141 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7201 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7261 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7321 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7381 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7441 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7501 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7561 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7621 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7681 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7741 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7921 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     8041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     8101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     8341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     8401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8761 ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata
     8821 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     8881 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     8941 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     9001 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     9061 gaaattgtat ttgttaaata tgtactacaa acttagtagt t
//
