LOCUS       sequence-430317-        8952 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 8952)
  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 8952)
  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: LMAPZW
FEATURES             Location/Qualifiers
     source          1..8952
                     /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"
     primer_bind     complement(4622..4641)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5096..5116)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5514..5525)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5715..6348
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6477..6493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6477..6493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6490..6512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6501..6517
                     /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(6525..6531,6532..6549,6550..6555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6570..6591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6708..6725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6879..7467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6959..6978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7638..8429,8430..8498))
                     /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     8261..8280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8499..8603)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8651..8785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8688..8707)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8742..8761
                     /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 cccgctgagc
     4081 aatggaagcg ggtgggcttt gggggcggcg gccaatagca gttttgctcc ttcgctttct
     4141 gggctcagag gcggggaggg ggggggtccg ggggcgggct caggggcggg ctcaggggcg
     4201 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     4261 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctttaat taaccaccat
     4321 gagcaaaggc gaagagctgt tcaccggcgt ggtgcccatc ctggtggagc tggacggcga
     4381 cgtgaacggc cacaagttca gcgtgagcgg cgagggcgag ggcgacgcca cctacggcaa
     4441 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctggt
     4501 gaccaccctg agccacggcg tgcagtgctt cagccggtac cccgaccaca tgaagcagca
     4561 cgacttcttc aagagcgcca tgcccgaggg ctacgtgcag gagcggacca tcttcttcaa
     4621 ggacgacggc aactacaaga cccgggccga ggtgaagttc gagggcgaca ccctggtgaa
     4681 ccggatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg gccacaagct
     4741 ggagtacaac ttcaacagcc acaacatcta catcatggcc gacaagcaga agaacggcat
     4801 caaggtgaac ttcaagatcc ggcacaacat cgaggacggc agcgtgcagc tggccgacca
     4861 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     4921 gagcacccag agcgccctga gcaaggaccc caacgagaag cgggaccaca tggtgctgct
     4981 ggagttccgg accgccgccg gcatcaccca cggcatggac gagctctata agtaagttta
     5041 aacatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     5101 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     5161 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     5221 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     5281 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     5341 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     5401 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     5461 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     5521 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     5581 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggcctgc
     5641 aggggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5701 agaaaagagg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5761 gtggatctac cacacacaag gctacttccc tgattagcag aactacacac cagggccagg
     5821 ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa
     5881 ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg
     5941 gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca
     6001 tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta
     6061 caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg
     6121 gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt
     6181 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     6241 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6301 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     6361 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc
     6421 cttgacattg ctagcgtttt accgtcgacc tctagctaga gcttggcgta atcatggtca
     6481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     8041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     8641 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     8701 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     8761 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     8821 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     8881 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     8941 aaacttagta gt
//
