LOCUS       sequence-409140-        8778 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 8778)
  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 8778)
  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: LHAPPW
FEATURES             Location/Qualifiers
     source          1..8778
                     /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..2396
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        3745..4244
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3780..3803)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4177..4196
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4262..4861
                     /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(4262..4281)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4758..4778
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4922..4942)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5340..5351)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5541..6174
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6303..6319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6303..6319)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6316..6338)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6327..6343
                     /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(6351..6357,6358..6375,6376..6381))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6396..6417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6534..6551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6705..7293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6785..6804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7464..8255,8256..8324))
                     /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     8087..8106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8325..8429)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8477..8611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8514..8533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8568..8587
                     /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 cgatgggcag agcgcacatc gcccacagtc cccgagaagt
     2221 tggggggagg ggtcggcaat tgaaccggag cctagagaag gtggcgcggg gtaaactggg
     2281 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2341 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagggct
     2401 cgcatctctc cttcacgcgc ccgccgcctt acctgaggcc gccatccacg ccggttgagt
     2461 cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaacta cgtccgccgt ctaggtaagt
     2521 ttagagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc
     2581 agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt
     2641 tctgttctgc gccgttacag atcagcgctt cgactaggga taacagggta attgtttgaa
     2701 tgaggcttca gtactttaca gaatcgttgc ctgcacatct tggaaacact tgctgggatt
     2761 acttcgactt cttaacccaa cagaaggctc gagaaggtat attgctgttg acaggatcca
     2821 cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat gatttcacta
     2881 atggttcaat ttttaatttc ttttcttctg gttcaaaatt caagtttcaa gtgaaagtgt
     2941 aatatgcacc catttctttc aataaatctt ctaaatagtc cactaatgtt ttattttgtt
     3001 ttttacaaaa tcaagcagcc tctgctatca atatagaacc ttgtattcca tctttatctc
     3061 tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca aaatttaatc
     3121 cgttatcttc ttctttagca atttctctac ccattcattt caatccagtt aaagttttta
     3181 caatcttaac tccatatttt tcctgagcga ttctatcacc caaatcactt gttacaaaac
     3241 ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt gataattttc
     3301 aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga ccatcatgtt
     3361 ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata tctgcatcat
     3421 gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga tttggattta
     3481 caacattttt caatgtttca tcttcaaatg cacgatcttc accctcaata acgttatatc
     3541 ctgattcacg taatcttttt ggggtaaatt tagttcctgt tccattaact gcgctaaaaa
     3601 tagggccctg cctcggactt caaggggcta gaattcgagc aattatcttg tttactaaaa
     3661 ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa tggtataaat
     3721 taaatcactt ttttcaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3781 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3841 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3901 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3961 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     4021 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     4081 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     4141 gcngggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     4201 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgaccttt aattaaccac
     4261 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     4321 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     4381 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     4441 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     4501 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     4561 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     4621 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     4681 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     4741 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     4801 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     4861 agtttaaaca tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     4921 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     4981 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     5041 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     5101 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     5161 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     5221 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     5281 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     5341 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     5401 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     5461 gcctgcaggg gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5521 tttaaaagaa aagaggggac tggaagggct aattcactcc caacgaagac aagatatcct
     5581 tgatctgtgg atctaccaca cacaaggcta cttccctgat tagcagaact acacaccagg
     5641 gccaggggtc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc
     5701 agataaggta gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct
     5761 gcatgggatg gatgacccgg agagagaagt gttagagtgg aggtttgaca gccgcctagc
     5821 atttcatcac gtggcccgag agctgcatcc ggagtacttc aagaactgct gatatcgagc
     5881 ttgctacaag ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg
     5941 ggagtggcga gccctcagat cctgcatata agcagctgct ttttgcctgt actgggtctc
     6001 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     6061 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     6121 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     6181 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     6241 agaggccttg acattgctag cgttttaccg tcgacctcta gctagagctt ggcgtaatca
     6301 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6361 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6421 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6481 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6541 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6601 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6661 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6721 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6781 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6841 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6901 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6961 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7021 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7081 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7141 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7201 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7261 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7321 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7381 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7441 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7501 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7561 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7621 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7681 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7741 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7801 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7861 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7921 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7981 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8041 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8101 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8161 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8221 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8281 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8341 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8401 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     8461 gacggatcgg gagatcaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     8521 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     8581 actcatcaat gtatcttatc atgtctggat caactggata actcaagcta accaaaatca
     8641 tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt ttcatttact
     8701 ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg
     8761 tactacaaac ttagtagt
//
