LOCUS       sequence-430235-        9218 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 9218)
  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 9218)
  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: LBAPZW
FEATURES             Location/Qualifiers
     source          1..9218
                     /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        2188..2473
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2475..2752
                     /label=chicken beta-actin promoter
     promoter        4068..4567
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4103..4126)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4500..4519
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(4888..4907)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5362..5382)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5780..5791)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5981..6614
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6743..6759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6743..6759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6756..6778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6767..6783
                     /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(6791..6797,6798..6815,6816..6821))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6836..6857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6974..6991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7145..7733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7225..7244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7904..8695,8696..8764))
                     /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     8527..8546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8765..8869)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8917..9051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8954..8973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9008..9027
                     /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 cgatagtcgt tacataactt acggtaaatg gcccgcctgg
     2221 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
     2281 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2341 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
     2401 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2461 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     2521 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     2581 gggggggggg ggggggcgcg cgccrggggg ggsggggsgg ggsgrggggs ggggsggggs
     2641 grggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     2701 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     2761 tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     2821 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     2881 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     2941 ttaattacct gttttacagg cctgaaatca cttggtttta ggttggagcg cttcgactag
     3001 ggataacagg gtaattgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca
     3061 tcttggaaac acttgctggg attacttcga cttcttaacc caacagaagg ctcgagaagg
     3121 tatattgctg ttgacaggat ccacattaac aactttaagt ccagcaattt gagttaaggg
     3181 tgttgctctc aatgatttca ctaatggttc aatttttaat ttcttttctt ctggttcaaa
     3241 attcaagttt caagtgaaag tgtaatatgc acccatttct ttcaataaat cttctaaata
     3301 gtccactaat gttttatttt gttttttaca aaatcaagca gcctctgcta tcaatataga
     3361 accttgtatt ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc
     3421 ataagcaaaa acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca
     3481 tttcaatcca gttaaagttt ttacaatctt aactccatat ttttcctgag cgattctatc
     3541 acccaaatca cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa
     3601 gcgttttaga tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa
     3661 tcttacaaaa tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt
     3721 cataataata atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg
     3781 atcaaattct ggatttggat ttacaacatt tttcaatgtt tcatcttcaa atgcacgatc
     3841 ttcaccctca ataacgttat atcctgattc acgtaatctt tttggggtaa atttagttcc
     3901 tgttccatta actgcgctaa aaatagggcc ctgcctcgga cttcaagggg ctagaattcg
     3961 agcaattatc ttgtttacta aaactgaata ccttgctatc tctttgatac atttttacaa
     4021 agctgaatta aaatggtata aattaaatca cttttttcaa ttctaccggg taggggaggc
     4081 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     4141 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     4201 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     4261 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     4321 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     4381 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
     4441 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
     4501 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
     4561 cctttcgacc tttaattaac caccatgagc aaaggcgaag agctgttcac cggcgtggtg
     4621 cccatcctgg tggagctgga cggcgacgtg aacggccaca agttcagcgt gagcggcgag
     4681 ggcgagggcg acgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     4741 ctgcccgtgc cctggcccac cctggtgacc accctgagcc acggcgtgca gtgcttcagc
     4801 cggtaccccg accacatgaa gcagcacgac ttcttcaaga gcgccatgcc cgagggctac
     4861 gtgcaggagc ggaccatctt cttcaaggac gacggcaact acaagacccg ggccgaggtg
     4921 aagttcgagg gcgacaccct ggtgaaccgg atcgagctga agggcatcga cttcaaggag
     4981 gacggcaaca tcctgggcca caagctggag tacaacttca acagccacaa catctacatc
     5041 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccggca caacatcgag
     5101 gacggcagcg tgcagctggc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     5161 gtgctgctgc ccgacaacca ctacctgagc acccagagcg ccctgagcaa ggaccccaac
     5221 gagaagcggg accacatggt gctgctggag ttccggaccg ccgccggcat cacccacggc
     5281 atggacgagc tctataagta agtttaaaca tcaacctctg gattacaaaa tttgtgaaag
     5341 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     5401 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     5461 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     5521 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     5581 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     5641 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     5701 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     5761 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     5821 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     5881 ttgggccgcc tccccgcctg gcctgcaggg gtacctttaa gaccaatgac ttacaaggca
     5941 gctgtagatc ttagccactt tttaaaagaa aagaggggac tggaagggct aattcactcc
     6001 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     6061 tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg atggtgctac
     6121 aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga gaacaccagc
     6181 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt gttagagtgg
     6241 aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc ggagtacttc
     6301 aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg
     6361 cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata agcagctgct
     6421 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     6481 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     6541 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6601 gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     6661 gaaatgaata tcagagagtg agaggccttg acattgctag cgttttaccg tcgacctcta
     6721 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6781 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6841 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6901 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6961 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     7021 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     7081 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     7141 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     7201 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     7261 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     7321 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     7381 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7441 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7501 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     7561 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     7621 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7681 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7741 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7801 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7861 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7921 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7981 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     8041 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     8101 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     8161 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     8221 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     8281 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     8341 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     8401 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     8461 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     8521 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     8581 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     8641 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8701 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8761 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8821 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8881 gaaaagtgcc acctgacgtc gacggatcgg gagatcaact tgtttattgc agcttataat
     8941 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     9001 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat caactggata
     9061 actcaagcta accaaaatca tcccaaactt cccaccccat accctattac cactgccaat
     9121 tacctgtggt ttcatttact ctaaacctgt gattcctctg aattattttc attttaaaga
     9181 aattgtattt gttaaatatg tactacaaac ttagtagt
//
