LOCUS       sequence-409026-        8835 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 8835)
  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 8835)
  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: LMAPPW
FEATURES             Location/Qualifiers
     source          1..8835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2185..2720
                     /label=mCMV promoter
                     /note="mouse cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        3802..4301
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3837..3860)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4234..4253
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4319..4918
                     /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(4319..4338)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4815..4835
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4979..4999)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5397..5408)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5598..6231
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6360..6376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6360..6376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6373..6395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6384..6400
                     /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(6408..6414,6415..6432,6433..6438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6453..6474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6591..6608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6762..7350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6842..6861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7521..8312,8313..8381))
                     /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     8144..8163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8382..8486)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8534..8668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8571..8590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8625..8644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgattggcac gtatactgag tcattaggga ctttccaatg
     2221 ggttttgccc agtacataag gtcaataggg gtgaatcaac aggaaagtcc cattggagcc
     2281 aagtacactg agtcaatagg gactttccat tgggttttgc ccagtacaaa aggtcaatag
     2341 ggggtgagtc aatgggtttt tcccattatt ggcacgtaca taaggtcaat aggggtgagt
     2401 cattgggttt ttccagccaa tttaattata acgccatgta ctttcccacc attgacgtca
     2461 atgggctatt gaaactaatg caacgtgacc tttaaacggt actttcccat agctgattaa
     2521 tgggaaagta ccgttgtcga gccaatacac gtcaatggga agtgaaaggg cagccaaaac
     2581 gtaacaccgc cccggttttc ccctggaaat tccatattgg cacgcattct attggctgag
     2641 ctgcgttcta cgtgggtata agaggcgcga ccagcgtccg taccgtcgca gtcttcggtc
     2701 tgaccaccgt agaacgcaga agcgcttcga ctagggataa cagggtaatt gtttgaatga
     2761 ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc tgggattact
     2821 tcgacttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca ggatccacat
     2881 taacaacttt aagtccagca atttgagtta agggtgttgc tctcaatgat ttcactaatg
     2941 gttcaatttt taatttcttt tcttctggtt caaaattcaa gtttcaagtg aaagtgtaat
     3001 atgcacccat ttctttcaat aaatcttcta aatagtccac taatgtttta ttttgttttt
     3061 tacaaaatca agcagcctct gctatcaata tagaaccttg tattccatct ttatctctag
     3121 ctgagtcatc aattacatat ccataacttt cttcataagc aaaaacaaaa tttaatccgt
     3181 tatcttcttc tttagcaatt tctctaccca ttcatttcaa tccagttaaa gtttttacaa
     3241 tcttaactcc atatttttcc tgagcgattc tatcacccaa atcacttgtt acaaaacttg
     3301 aatatagagc cggatttttt ggaatgctat ttaagcgttt tagatttgat aattttcaat
     3361 caattaaaat tggtcctgtt tgatttccat ctaatcttac aaaatgacca tcatgtttta
     3421 ttgccattcc aaatctgtca gcatctgggt cattcataat aataatatct gcatcatgtt
     3481 taataccata ttcaagcggt atttttcatg caggatcaaa ttctggattt ggatttacaa
     3541 catttttcaa tgtttcatct tcaaatgcac gatcttcacc ctcaataacg ttatatcctg
     3601 attcacgtaa tctttttggg gtaaatttag ttcctgttcc attaactgcg ctaaaaatag
     3661 ggccctgcct cggacttcaa ggggctagaa ttcgagcaat tatcttgttt actaaaactg
     3721 aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg tataaattaa
     3781 atcacttttt tcaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     3841 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     3901 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     3961 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     4021 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     4081 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     4141 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     4201 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     4261 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctttaat taaccaccat
     4321 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     4381 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     4441 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     4501 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     4561 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     4621 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     4681 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     4741 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     4801 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     4861 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagt
     4921 ttaaacatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     4981 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5041 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5101 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5161 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5221 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5281 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     5341 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     5401 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     5461 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggcc
     5521 tgcaggggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     5581 aaaagaaaag aggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     5641 tctgtggatc taccacacac aaggctactt ccctgattag cagaactaca caccagggcc
     5701 aggggtcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagccaga
     5761 taaggtagaa gaggccaata aaggagagaa caccagcttg ttacaccctg tgagcctgca
     5821 tgggatggat gacccggaga gagaagtgtt agagtggagg tttgacagcc gcctagcatt
     5881 tcatcacgtg gcccgagagc tgcatccgga gtacttcaag aactgctgat atcgagcttg
     5941 ctacaaggga ctttccgctg gggactttcc agggaggcgt ggcctgggcg ggactgggga
     6001 gtggcgagcc ctcagatcct gcatataagc agctgctttt tgcctgtact gggtctctct
     6061 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     6121 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     6181 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     6241 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     6301 ggccttgaca ttgctagcgt tttaccgtcg acctctagct agagcttggc gtaatcatgg
     6361 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6421 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6481 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6541 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6601 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6661 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6721 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6781 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6841 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6901 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6961 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7021 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7081 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7141 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7201 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7261 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7321 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7381 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7441 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7501 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7561 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7621 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7681 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7741 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7801 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7861 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7921 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7981 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8041 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8101 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8161 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8221 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8281 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8341 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8401 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8461 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     8521 ggatcgggag atcaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     8581 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     8641 catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc
     8701 caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta
     8761 aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac
     8821 tacaaactta gtagt
//
