LOCUS       sequence-430338-        8913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with E2-Crimson reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8913)
  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 8913)
  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: LMAPEW
FEATURES             Location/Qualifiers
     source          1..8913
                     /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"
     CDS             4319..4996
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(5057..5077)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5475..5486)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5676..6309
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6438..6454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6438..6454)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6451..6473)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6462..6478
                     /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(6486..6492,6493..6510,6511..6516))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6531..6552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6669..6686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6840..7428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6920..6939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7599..8390,8391..8459))
                     /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     8222..8241
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8460..8564)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8612..8746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8649..8668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8703..8722
                     /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 ggatagcact gagaacgtca tcaagccctt catgcgcttc aaggtgcaca tggagggctc
     4381 cgtgaacggc cacgagttcg agatcgaggg cgtgggcgag ggcaagccct acgagggcac
     4441 ccagaccgcc aagctgcaag tgaccaaggg cggccccctg cccttcgcct gggacatcct
     4501 gtccccccag ttcttctacg gctccaaggc gtacatcaag caccccgccg acatccccga
     4561 ctacctcaag cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     4621 cggcggcgtg gtgaccgtga cccaggactc atccctgcaa gacggcaccc tcatctacca
     4681 cgtcaagttc atcggcgtga acttcccctc cgacggcccc gtaatgcaga agaaaactct
     4741 tggctgggag ccctccactg agcgcaacta cccccgcgac ggcgtgctga agggcgagaa
     4801 ccacatggcg ctgaagctga agggcggcgg ccactacctg tgtgagttca agtccatcta
     4861 catggctaag aagcccgtga agctgcccgg ctaccactac gtggactaca agctcgacat
     4921 cacctcccac aacgaggact acaccgtggt ggagcagtac gaacgcgccg aggcccgcca
     4981 ccacctgttc cagtaggttt aaacatcaac ctctggatta caaaatttgt gaaagattga
     5041 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5101 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5161 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5221 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5281 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5341 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc
     5401 tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5461 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     5521 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     5581 ccgcctcccc gcctggcctg caggggtacc tttaagacca atgacttaca aggcagctgt
     5641 agatcttagc cactttttaa aagaaaagag gggactggaa gggctaattc actcccaacg
     5701 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattagca
     5761 gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt gctacaagct
     5821 agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca ccagcttgtt
     5881 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag agtggaggtt
     5941 tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt acttcaagaa
     6001 ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg
     6061 cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag ctgctttttg
     6121 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6181 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6241 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6301 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     6361 gaatatcaga gagtgagagg ccttgacatt gctagcgttt taccgtcgac ctctagctag
     6421 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6481 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6541 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6601 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6661 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6721 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6781 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6841 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6901 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6961 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     7021 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     7081 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7141 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7201 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7261 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7321 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7381 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7441 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7501 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7561 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7621 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7681 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7741 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7801 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7861 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7921 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7981 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     8041 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     8101 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8161 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8221 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8281 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8341 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8401 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8461 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     8521 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     8581 gtgccacctg acgtcgacgg atcgggagat caacttgttt attgcagctt ataatggtta
     8641 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     8701 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca
     8761 agctaaccaa aatcatccca aacttcccac cccataccct attaccactg ccaattacct
     8821 gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg
     8881 tatttgttaa atatgtacta caaacttagt agt
//
