LOCUS       sequence-409380-        9027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with Azurite reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9027)
  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 9027)
  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: LCAPZW
FEATURES             Location/Qualifiers
     source          1..9027
                     /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)"
     enhancer        2201..2504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2505..2708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2659..2679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2705..2729
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3877..4376
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3912..3935)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4309..4328
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(4697..4716)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5171..5191)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5589..5600)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5790..6423
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6552..6568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6552..6568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6565..6587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6576..6592
                     /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(6600..6606,6607..6624,6625..6630))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6645..6666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6783..6800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6954..7542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7034..7053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7713..8504,8505..8573))
                     /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     8336..8355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8574..8678)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8726..8860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8763..8782)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8817..8836
                     /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 cgataagctt gggagttccg cgttacataa cttacggtaa
     2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     2761 tccatagaag acaccgactc tactagagga tcgctagcgc ttcgactagg gataacaggg
     2821 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     2881 cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt atattgctgt
     2941 tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt gttgctctca
     3001 atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa ttcaagtttc
     3061 aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag tccactaatg
     3121 ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa ccttgtattc
     3181 catctttatc tctagctgag tcatcaatta catatccata actttcttca taagcaaaaa
     3241 caaaatttaa tccgttatct tcttctttag caatttctct acccattcat ttcaatccag
     3301 ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca cccaaatcac
     3361 ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag cgttttagat
     3421 ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat cttacaaaat
     3481 gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc ataataataa
     3541 tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga tcaaattctg
     3601 gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct tcaccctcaa
     3661 taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct gttccattaa
     3721 ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga gcaattatct
     3781 tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa
     3841 aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg cttttcccaa
     3901 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     3961 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     4021 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     4081 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     4141 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     4201 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     4261 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     4321 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     4381 ttaattaacc accatgagca aaggcgaaga gctgttcacc ggcgtggtgc ccatcctggt
     4441 ggagctggac ggcgacgtga acggccacaa gttcagcgtg agcggcgagg gcgagggcga
     4501 cgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     4561 ctggcccacc ctggtgacca ccctgagcca cggcgtgcag tgcttcagcc ggtaccccga
     4621 ccacatgaag cagcacgact tcttcaagag cgccatgccc gagggctacg tgcaggagcg
     4681 gaccatcttc ttcaaggacg acggcaacta caagacccgg gccgaggtga agttcgaggg
     4741 cgacaccctg gtgaaccgga tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     4801 cctgggccac aagctggagt acaacttcaa cagccacaac atctacatca tggccgacaa
     4861 gcagaagaac ggcatcaagg tgaacttcaa gatccggcac aacatcgagg acggcagcgt
     4921 gcagctggcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4981 cgacaaccac tacctgagca cccagagcgc cctgagcaag gaccccaacg agaagcggga
     5041 ccacatggtg ctgctggagt tccggaccgc cgccggcatc acccacggca tggacgagct
     5101 ctataagtaa gtttaaacat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     5161 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5221 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5281 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5341 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5401 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5461 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     5521 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     5581 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     5641 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     5701 ccccgcctgg cctgcagggg tacctttaag accaatgact tacaaggcag ctgtagatct
     5761 tagccacttt ttaaaagaaa agaggggact ggaagggcta attcactccc aacgaagaca
     5821 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt agcagaacta
     5881 cacaccaggg ccaggggtca gatatccact gacctttgga tggtgctaca agctagtacc
     5941 agttgagcca gataaggtag aagaggccaa taaaggagag aacaccagct tgttacaccc
     6001 tgtgagcctg catgggatgg atgacccgga gagagaagtg ttagagtgga ggtttgacag
     6061 ccgcctagca tttcatcacg tggcccgaga gctgcatccg gagtacttca agaactgctg
     6121 atatcgagct tgctacaagg gactttccgc tggggacttt ccagggaggc gtggcctggg
     6181 cgggactggg gagtggcgag ccctcagatc ctgcatataa gcagctgctt tttgcctgta
     6241 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     6301 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     6361 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     6421 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     6481 cagagagtga gaggccttga cattgctagc gttttaccgt cgacctctag ctagagcttg
     6541 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     6601 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     6661 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6721 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6781 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6841 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6901 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6961 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     7021 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     7081 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     7141 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     7201 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     7261 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     7321 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     7381 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7441 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7501 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7561 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7621 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7681 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7741 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7801 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7861 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7921 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7981 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     8041 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     8101 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     8161 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     8221 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     8281 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     8341 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     8401 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8461 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8521 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     8581 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8641 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8701 cctgacgtcg acggatcggg agatcaactt gtttattgca gcttataatg gttacaaata
     8761 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     8821 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa
     8881 ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt
     8941 tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg
     9001 ttaaatatgt actacaaact tagtagt
//
