LOCUS       sequence-430325-        8898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with Ametrine reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8898)
  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 8898)
  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: LHAPAW
FEATURES             Location/Qualifiers
     source          1..8898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    680..805
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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    2027..2142
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2184..2395
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        3744..4243
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3779..3802)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4176..4195
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(4261..4263,4264..4266,4267..4980)
                     /codon_start=1
                     /product="photostable yellow fluorescent protein with a 
                     large Stokes shift, excitable with violet light (Ai et al.,
                     2008)"
                     /label=mAmetrine1.1
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVRGEGEGDATNGKLTL
                     KFICTSGELPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNAWDAYITADKQKNGIK
                     ANFKIEHNVKDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(4306..4327)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5041..5061)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5459..5470)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5660..6293
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6422..6438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6422..6438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6435..6457)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6446..6462
                     /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(6470..6476,6477..6494,6495..6500))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6515..6536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6653..6670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6824..7412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6904..6923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7583..8374,8375..8443))
                     /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     8206..8225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8444..8548)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8596..8730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8633..8652)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8687..8706
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(8898,1..633)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac
       61 acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact
      121 gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa
      181 taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga
      241 gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga
      301 gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc
      361 tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc
      421 ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag
      481 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      541 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      601 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      661 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      721 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      781 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      841 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      901 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      961 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1021 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1081 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1141 agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1981 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccttta aaagaaaagg
     2041 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2101 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2161 cagcagagat ccagtttatc gatgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2221 ggggggaggg gtcggcaatt gaaccggagc ctagagaagg tggcgcgggg taaactggga
     2281 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2341 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagggctc
     2401 gcatctctcc ttcacgcgcc cgccgcctta cctgaggccg ccatccacgc cggttgagtc
     2461 gcgttctgcc gcctcccgcc tgtggtgcct cctgaactac gtccgccgtc taggtaagtt
     2521 tagagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca
     2581 gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt
     2641 ctgttctgcg ccgttacaga tcagcgcttc gactagggat aacagggtaa ttgtttgaat
     2701 gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta
     2761 cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga caggatccac
     2821 attaacaact ttaagtccag caatttgagt taagggtgtt gctctcaatg atttcactaa
     2881 tggttcaatt tttaatttct tttcttctgg ttcaaaattc aagtttcaag tgaaagtgta
     2941 atatgcaccc atttctttca ataaatcttc taaatagtcc actaatgttt tattttgttt
     3001 tttacaaaat caagcagcct ctgctatcaa tatagaacct tgtattccat ctttatctct
     3061 agctgagtca tcaattacat atccataact ttcttcataa gcaaaaacaa aatttaatcc
     3121 gttatcttct tctttagcaa tttctctacc cattcatttc aatccagtta aagtttttac
     3181 aatcttaact ccatattttt cctgagcgat tctatcaccc aaatcacttg ttacaaaact
     3241 tgaatataga gccggatttt ttggaatgct atttaagcgt tttagatttg ataattttca
     3301 atcaattaaa attggtcctg tttgatttcc atctaatctt acaaaatgac catcatgttt
     3361 tattgccatt ccaaatctgt cagcatctgg gtcattcata ataataatat ctgcatcatg
     3421 tttaatacca tattcaagcg gtatttttca tgcaggatca aattctggat ttggatttac
     3481 aacatttttc aatgtttcat cttcaaatgc acgatcttca ccctcaataa cgttatatcc
     3541 tgattcacgt aatctttttg gggtaaattt agttcctgtt ccattaactg cgctaaaaat
     3601 agggccctgc ctcggacttc aaggggctag aattcgagca attatcttgt ttactaaaac
     3661 tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaatt
     3721 aaatcacttt tttcaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3781 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     3841 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     3901 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     3961 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     4021 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     4081 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     4141 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     4201 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgaccttta attaaccacc
     4261 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4321 ggcgacgtaa acggccacag gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac
     4381 ggcaagctga ccctgaagtt catctgcacc tccggcgagc tgcccgtgcc ctggcccacc
     4441 ctcgtgacca ccctgtctta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     4501 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc
     4561 ttcaaggacg acggcagcta caggacccgc gccgaggtga agttcgaggg cgacaccctg
     4621 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4681 aagctggagt acaacatgaa cgcttgggac gcttatatca cggccgacaa gcagaagaac
     4741 ggcatcaaag cgaacttcaa gatcgagcac aacgtcaagg acggcggcgt gcagctcgcc
     4801 gacgcttacc agcagaacac ccccatcggc gacggctccg tgctgctgcc tgacaaccac
     4861 tacctgagct tccagagcaa gctgttcaaa gaccccaacg agcagcgcga tcacatggtc
     4921 ctgctggagt tcgttaccgc cgccgggata actcccggca tggacgagct gtacaagtaa
     4981 gtttaaacat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     5041 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     5101 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     5161 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     5221 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     5281 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     5341 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     5401 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     5461 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5521 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
     5581 cctgcagggg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5641 ttaaaagaaa agaggggact ggaagggcta attcactccc aacgaagaca agatatcctt
     5701 gatctgtgga tctaccacac acaaggctac ttccctgatt agcagaacta cacaccaggg
     5761 ccaggggtca gatatccact gacctttgga tggtgctaca agctagtacc agttgagcca
     5821 gataaggtag aagaggccaa taaaggagag aacaccagct tgttacaccc tgtgagcctg
     5881 catgggatgg atgacccgga gagagaagtg ttagagtgga ggtttgacag ccgcctagca
     5941 tttcatcacg tggcccgaga gctgcatccg gagtacttca agaactgctg atatcgagct
     6001 tgctacaagg gactttccgc tggggacttt ccagggaggc gtggcctggg cgggactggg
     6061 gagtggcgag ccctcagatc ctgcatataa gcagctgctt tttgcctgta ctgggtctct
     6121 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     6181 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     6241 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     6301 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     6361 gaggccttga cattgctagc gttttaccgt cgacctctag ctagagcttg gcgtaatcat
     6421 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6481 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6541 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6601 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6661 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6721 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6781 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6841 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6901 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6961 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7021 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7081 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7141 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7201 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7261 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7321 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7381 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7441 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7501 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7561 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7621 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7681 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7741 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7801 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7861 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7921 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7981 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     8041 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     8101 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     8161 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     8221 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8281 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8341 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8401 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8461 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8521 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     8581 acggatcggg agatcaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     8641 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     8701 ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa ccaaaatcat
     8761 cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt tcatttactc
     8821 taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg ttaaatatgt
     8881 actacaaact tagtagtt
//
