LOCUS       sequence-409113-        8895 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 8895)
  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 8895)
  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: LHAPZW
FEATURES             Location/Qualifiers
     source          1..8895
                     /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"
     primer_bind     complement(4565..4584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5039..5059)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5457..5468)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5658..6291
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6420..6436)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6420..6436)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6433..6455)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6444..6460
                     /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(6468..6474,6475..6492,6493..6498))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6513..6534
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6651..6668)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6822..7410)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6902..6921)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7581..8372,8373..8441))
                     /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     8204..8223
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8442..8546)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8594..8728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8631..8650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8685..8704
                     /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 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     4201 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgaccttt aattaaccac
     4261 catgagcaaa ggcgaagagc tgttcaccgg cgtggtgccc atcctggtgg agctggacgg
     4321 cgacgtgaac ggccacaagt tcagcgtgag cggcgagggc gagggcgacg ccacctacgg
     4381 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     4441 ggtgaccacc ctgagccacg gcgtgcagtg cttcagccgg taccccgacc acatgaagca
     4501 gcacgacttc ttcaagagcg ccatgcccga gggctacgtg caggagcgga ccatcttctt
     4561 caaggacgac ggcaactaca agacccgggc cgaggtgaag ttcgagggcg acaccctggt
     4621 gaaccggatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tgggccacaa
     4681 gctggagtac aacttcaaca gccacaacat ctacatcatg gccgacaagc agaagaacgg
     4741 catcaaggtg aacttcaaga tccggcacaa catcgaggac ggcagcgtgc agctggccga
     4801 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4861 cctgagcacc cagagcgccc tgagcaagga ccccaacgag aagcgggacc acatggtgct
     4921 gctggagttc cggaccgccg ccggcatcac ccacggcatg gacgagctct ataagtaagt
     4981 ttaaacatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5041 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5101 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5161 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5221 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5281 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5341 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     5401 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     5461 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     5521 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggcc
     5581 tgcaggggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     5641 aaaagaaaag aggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     5701 tctgtggatc taccacacac aaggctactt ccctgattag cagaactaca caccagggcc
     5761 aggggtcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagccaga
     5821 taaggtagaa gaggccaata aaggagagaa caccagcttg ttacaccctg tgagcctgca
     5881 tgggatggat gacccggaga gagaagtgtt agagtggagg tttgacagcc gcctagcatt
     5941 tcatcacgtg gcccgagagc tgcatccgga gtacttcaag aactgctgat atcgagcttg
     6001 ctacaaggga ctttccgctg gggactttcc agggaggcgt ggcctgggcg ggactgggga
     6061 gtggcgagcc ctcagatcct gcatataagc agctgctttt tgcctgtact gggtctctct
     6121 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     6181 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     6241 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     6301 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     6361 ggccttgaca ttgctagcgt tttaccgtcg acctctagct agagcttggc gtaatcatgg
     6421 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6481 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6541 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6601 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6661 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6721 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6781 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6841 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6901 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6961 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     7021 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7081 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7141 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7201 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7261 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7321 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7381 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7441 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7501 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7561 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7621 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7681 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7741 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7801 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7861 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7921 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7981 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     8041 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8101 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8161 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8221 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8281 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8341 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8401 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8461 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8521 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     8581 ggatcgggag atcaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     8641 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     8701 catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc
     8761 caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta
     8821 aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac
     8881 tacaaactta gtagt
//
