LOCUS       sequence-430236-        9580 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 9580)
  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 9580)
  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: LEAPZW
FEATURES             Location/Qualifiers
     source          1..9580
                     /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"
     intron          2397..3339
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3296..3316
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        4430..4929
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4465..4488)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4862..4881
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(5250..5269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5724..5744)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6142..6153)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6343..6976
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(7105..7121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7105..7121)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7118..7140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7129..7145
                     /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(7153..7159,7160..7177,7178..7183))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7198..7219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7336..7353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7507..8095)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7587..7606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8266..9057,9058..9126))
                     /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     8889..8908
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9127..9231)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    9279..9413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9316..9335)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9370..9389
                     /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 acacaggtaa
     2401 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     2461 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2521 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2581 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2641 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2701 tttttctggc aagatagtct tgtaaatgcg ggccaagatc ggcacactgg tatttcggtt
     2761 tttggggccg cgggcggcga cggggccggt gcgtcccagc gcacatgttc ggcgaggcgg
     2821 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2881 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2941 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3001 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3061 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3121 cacctcgatt agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3181 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3241 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3301 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaag cgcttcgact
     3361 agggataaca gggtaattgt ttgaatgagg cttcagtact ttacagaatc gttgcctgca
     3421 catcttggaa acacttgctg ggattacttc gacttcttaa cccaacagaa ggctcgagaa
     3481 ggtatattgc tgttgacagg atccacatta acaactttaa gtccagcaat ttgagttaag
     3541 ggtgttgctc tcaatgattt cactaatggt tcaattttta atttcttttc ttctggttca
     3601 aaattcaagt ttcaagtgaa agtgtaatat gcacccattt ctttcaataa atcttctaaa
     3661 tagtccacta atgttttatt ttgtttttta caaaatcaag cagcctctgc tatcaatata
     3721 gaaccttgta ttccatcttt atctctagct gagtcatcaa ttacatatcc ataactttct
     3781 tcataagcaa aaacaaaatt taatccgtta tcttcttctt tagcaatttc tctacccatt
     3841 catttcaatc cagttaaagt ttttacaatc ttaactccat atttttcctg agcgattcta
     3901 tcacccaaat cacttgttac aaaacttgaa tatagagccg gattttttgg aatgctattt
     3961 aagcgtttta gatttgataa ttttcaatca attaaaattg gtcctgtttg atttccatct
     4021 aatcttacaa aatgaccatc atgttttatt gccattccaa atctgtcagc atctgggtca
     4081 ttcataataa taatatctgc atcatgttta ataccatatt caagcggtat ttttcatgca
     4141 ggatcaaatt ctggatttgg atttacaaca tttttcaatg tttcatcttc aaatgcacga
     4201 tcttcaccct caataacgtt atatcctgat tcacgtaatc tttttggggt aaatttagtt
     4261 cctgttccat taactgcgct aaaaataggg ccctgcctcg gacttcaagg ggctagaatt
     4321 cgagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat acatttttac
     4381 aaagctgaat taaaatggta taaattaaat cacttttttc aattctaccg ggtaggggag
     4441 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     4501 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     4561 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     4621 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     4681 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     4741 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     4801 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaagggcctc cggaggcccg
     4861 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     4921 ggcctttcga cctttaatta accaccatga gcaaaggcga agagctgttc accggcgtgg
     4981 tgcccatcct ggtggagctg gacggcgacg tgaacggcca caagttcagc gtgagcggcg
     5041 agggcgaggg cgacgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     5101 agctgcccgt gccctggccc accctggtga ccaccctgag ccacggcgtg cagtgcttca
     5161 gccggtaccc cgaccacatg aagcagcacg acttcttcaa gagcgccatg cccgagggct
     5221 acgtgcagga gcggaccatc ttcttcaagg acgacggcaa ctacaagacc cgggccgagg
     5281 tgaagttcga gggcgacacc ctggtgaacc ggatcgagct gaagggcatc gacttcaagg
     5341 aggacggcaa catcctgggc cacaagctgg agtacaactt caacagccac aacatctaca
     5401 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgg cacaacatcg
     5461 aggacggcag cgtgcagctg gccgaccact accagcagaa cacccccatc ggcgacggcc
     5521 ccgtgctgct gcccgacaac cactacctga gcacccagag cgccctgagc aaggacccca
     5581 acgagaagcg ggaccacatg gtgctgctgg agttccggac cgccgccggc atcacccacg
     5641 gcatggacga gctctataag taagtttaaa catcaacctc tggattacaa aatttgtgaa
     5701 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     5761 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     5821 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     5881 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     5941 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     6001 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     6061 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg
     6121 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     6181 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     6241 ctttgggccg cctccccgcc tggcctgcag gggtaccttt aagaccaatg acttacaagg
     6301 cagctgtaga tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact
     6361 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     6421 attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct
     6481 acaagctagt accagttgag ccagataagg tagaagaggc caataaagga gagaacacca
     6541 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt
     6601 ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact
     6661 tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga
     6721 ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg
     6781 ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     6841 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     6901 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     6961 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     7021 aagaaatgaa tatcagagag tgagaggcct tgacattgct agcgttttac cgtcgacctc
     7081 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     7141 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     7201 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     7261 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     7321 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     7381 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     7441 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     7501 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7561 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7621 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7681 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7741 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7801 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7861 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7921 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7981 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     8041 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     8101 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     8161 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     8221 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     8281 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     8341 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     8401 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     8461 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     8521 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     8581 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8641 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8701 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8761 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     8821 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     8881 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8941 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     9001 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     9061 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     9121 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     9181 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     9241 ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata
     9301 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     9361 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     9421 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     9481 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     9541 gaaattgtat ttgttaaata tgtactacaa acttagtagt
//
