LOCUS       sequence-409385-        9228 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 9228)
  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 9228)
  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: LBAPAW
FEATURES             Location/Qualifiers
     source          1..9228
                     /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)"
     enhancer        2187..2472
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2474..2751
                     /label=chicken beta-actin promoter
     promoter        4067..4566
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4102..4125)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4499..4518
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(4584..4586,4587..4589,4590..5303)
                     /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(4629..4650)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5371..5391)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5789..5800)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5990..6623
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6752..6768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6752..6768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6765..6787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6776..6792
                     /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(6800..6806,6807..6824,6825..6830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6845..6866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6983..7000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7154..7742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7234..7253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7913..8704,8705..8773))
                     /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     8536..8555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8774..8878)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8926..9060
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8963..8982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9017..9036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(9228,1..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 gatagtcgtt acataactta cggtaaatgg cccgcctggc
     2221 tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat
     2281 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2341 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt
     2401 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2461 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2521 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2581 gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
     2641 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2701 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2761 gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     2821 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     2881 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     2941 taattacctg ttttacaggc ctgaaatcac ttggttttag gttggagcgc ttcgactagg
     3001 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     3061 cttggaaaca cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt
     3121 atattgctgt tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt
     3181 gttgctctca atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa
     3241 ttcaagtttc aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag
     3301 tccactaatg ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa
     3361 ccttgtattc catctttatc tctagctgag tcatcaatta catatccata actttcttca
     3421 taagcaaaaa caaaatttaa tccgttatct tcttctttag caatttctct acccattcat
     3481 ttcaatccag ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca
     3541 cccaaatcac ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag
     3601 cgttttagat ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat
     3661 cttacaaaat gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc
     3721 ataataataa tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga
     3781 tcaaattctg gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct
     3841 tcaccctcaa taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct
     3901 gttccattaa ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga
     3961 gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa
     4021 gctgaattaa aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg
     4081 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     4141 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
     4201 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     4261 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
     4321 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     4381 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     4441 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     4501 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     4561 ctttcgacct ttaattaacc accatggtga gcaagggcga ggagctgttc accggggtgg
     4621 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgcgcggcg
     4681 agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc acctccggcg
     4741 agctgcccgt gccctggccc accctcgtga ccaccctgtc ttacggcgtg cagtgcttcg
     4801 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     4861 acgtccagga gcgcaccatc tccttcaagg acgacggcag ctacaggacc cgcgccgagg
     4921 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     4981 aggacggcaa catcctgggg cacaagctgg agtacaacat gaacgcttgg gacgcttata
     5041 tcacggccga caagcagaag aacggcatca aagcgaactt caagatcgag cacaacgtca
     5101 aggacggcgg cgtgcagctc gccgacgctt accagcagaa cacccccatc ggcgacggct
     5161 ccgtgctgct gcctgacaac cactacctga gcttccagag caagctgttc aaagacccca
     5221 acgagcagcg cgatcacatg gtcctgctgg agttcgttac cgccgccggg ataactcccg
     5281 gcatggacga gctgtacaag taagtttaaa caataaacat caacctctgg attacaaaat
     5341 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     5401 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     5461 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     5521 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     5581 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     5641 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5701 gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct
     5761 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5821 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5881 gatctccctt tgggccgcct ccccgcctgg cctgcagggg tacctttaag accaatgact
     5941 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa agaggggact ggaagggcta
     6001 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     6061 ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga
     6121 tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag
     6181 aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg
     6241 ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg
     6301 gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt
     6361 ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa
     6421 gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6481 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6541 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6601 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     6661 acttgcaaag aaatgaatat cagagagtga gaggccttga cattgctagc gttttaccgt
     6721 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6781 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6841 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6901 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6961 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     7021 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     7081 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7141 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7201 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7261 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7321 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7381 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7441 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7501 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7561 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7621 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7681 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7741 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7801 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7861 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7921 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7981 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     8041 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8101 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8161 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8221 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8281 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8341 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8401 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8461 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8521 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8581 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8641 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8701 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8761 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8821 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8881 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcaactt gtttattgca
     8941 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     9001 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     9061 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc
     9121 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca
     9181 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtt
//
