LOCUS       sequence-430322-        9212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with mOrange reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9212)
  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 9212)
  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: LBAPOW
FEATURES             Location/Qualifiers
     source          1..9212
                     /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        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"
     primer_bind     complement(5355..5375)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5773..5784)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5974..6607
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6736..6752)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6736..6752)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6749..6771)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6760..6776
                     /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(6784..6790,6791..6808,6809..6814))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6829..6850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6967..6984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7138..7726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7218..7237)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7897..8688,8689..8757))
                     /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     8520..8539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8758..8862)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8910..9044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8947..8966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9001..9020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(9212,1..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 gccrggsggg gsggggsggg gsgrggggsg gggsggggsg
     2641 rggsggrgag gkgcggcggc 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 agagaataac atggcaatca
     4621 tcaaggagtt catgcgcttc aaggttcgca tggagggctc cgtgaacggc cacgagttcg
     4681 agatcgaggg cgagggcgag ggccgcccct acgagggctt tcagaccgct aagctgaagg
     4741 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcacctacg
     4801 gctccaaggc atacgtgaag caccccgccg acatccccga ctacttcaag ctgtccttcc
     4861 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     4921 cccaggactc gtccctgcaa gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     4981 acttcccctc cgacggcccc gtaatgcaga aaaagactat gggctgggag gcatcctccg
     5041 agcggatgta ccccgaggac ggcgccctga agggcgagat caagatgagg ctgaagctga
     5101 aggacggcgg ccactacacc tccgaggtca agaccaccta caaggccaag aagcccgtgc
     5161 aactgcccgg cgcctacatc gtcggcatca agttggacat cacctcccac aacgaggact
     5221 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     5281 agctatacaa gtaggtttaa acatcaacct ctggattaca aaatttgtga aagattgact
     5341 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5401 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5461 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     5521 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5581 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5641 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     5701 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5761 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5821 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5881 gcctccccgc ctggcctgca ggggtacctt taagaccaat gacttacaag gcagctgtag
     5941 atcttagcca ctttttaaaa gaaaagaggg gactggaagg gctaattcac tcccaacgaa
     6001 gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga
     6061 actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag
     6121 taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac
     6181 accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg
     6241 acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact
     6301 gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc
     6361 tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc
     6421 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6481 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6541 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6601 tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     6661 atatcagaga gtgagaggcc ttgacattgc tagcgtttta ccgtcgacct ctagctagag
     6721 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6781 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6841 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6901 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6961 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7021 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     7081 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     7141 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     7201 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     7261 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     7321 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     7381 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     7441 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7501 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7561 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7621 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7681 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7741 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7801 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7861 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7921 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7981 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     8041 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     8101 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     8161 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     8221 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8281 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8341 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8401 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8461 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8521 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8581 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8641 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8701 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8761 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8821 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8881 gccacctgac gtcgacggat cgggagatca acttgtttat tgcagcttat aatggttaca
     8941 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     9001 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg ataactcaag
     9061 ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc aattacctgt
     9121 ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa agaaattgta
     9181 tttgttaaat atgtactaca aacttagtag tt
//
