LOCUS       sequence-409376-        8946 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 8946)
  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 8946)
  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: LMAPOW
FEATURES             Location/Qualifiers
     source          1..8946
                     /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(5090..5110)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5508..5519)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5709..6342
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6471..6487)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6471..6487)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6484..6506)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6495..6511
                     /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(6519..6525,6526..6543,6544..6549))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6564..6585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6702..6719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6873..7461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6953..6972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7632..8423,8424..8492))
                     /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     8255..8274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8493..8597)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8645..8779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8682..8701)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8736..8755
                     /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 accgctgagc
     4081 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     4141 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     4201 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     4261 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctttaat taaccaccat
     4321 ggtgagcaag ggcgaagaga ataacatggc aatcatcaag gagttcatgc gcttcaaggt
     4381 tcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     4441 cccctacgag ggctttcaga ccgctaagct gaaggtgacc aagggtggcc ccctgccctt
     4501 cgcctgggac atcctgtccc ctcagttcac ctacggctcc aaggcatacg tgaagcaccc
     4561 cgccgacatc cccgactact tcaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     4621 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcgtccc tgcaagacgg
     4681 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     4741 gcagaaaaag actatgggct gggaggcatc ctccgagcgg atgtaccccg aggacggcgc
     4801 cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact acacctccga
     4861 ggtcaagacc acctacaagg ccaagaagcc cgtgcaactg cccggcgcct acatcgtcgg
     4921 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     4981 cgccgagggc cgccactcca ccggcggcat ggacgagcta tacaagtagg tttaaacatc
     5041 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     5101 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     5161 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     5221 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     5281 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     5341 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     5401 gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct
     5461 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     5521 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     5581 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctggc ctgcaggggt
     5641 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     5701 gaggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat
     5761 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag
     5821 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga
     5881 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga
     5941 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt
     6001 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg
     6061 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc
     6121 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
     6181 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6241 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6301 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     6361 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggccttgac
     6421 attgctagcg ttttaccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     6481 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6541 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6601 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6661 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6721 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6781 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6841 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6901 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6961 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     7021 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     7081 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     7141 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     7201 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     7261 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     7321 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     7381 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     7441 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     7501 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     7561 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7621 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7681 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7741 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7801 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7861 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7921 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7981 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     8041 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     8101 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     8161 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     8221 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     8281 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     8341 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     8401 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     8461 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     8521 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     8581 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     8641 gatcaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     8701 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     8761 atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc
     8821 caccccatac cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga
     8881 ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt
     8941 agtagt
//
