LOCUS       sequence-409391-        9179 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 9179)
  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 9179)
  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: LBAPEW
FEATURES             Location/Qualifiers
     source          1..9179
                     /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             4584..5261
                     /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(5322..5342)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5740..5751)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5941..6574
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6703..6719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6703..6719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6716..6738)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6727..6743
                     /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(6751..6757,6758..6775,6776..6781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6796..6817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6934..6951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7105..7693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7185..7204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7864..8655,8656..8724))
                     /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     8487..8506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8725..8829)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8877..9011
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8914..8933)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8968..8987
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(9179,1..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 accatggata gcactgagaa cgtcatcaag cccttcatgc
     4621 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgtgg
     4681 gcgagggcaa gccctacgag ggcacccaga ccgccaagct gcaagtgacc aagggcggcc
     4741 ccctgccctt cgcctgggac atcctgtccc cccagttctt ctacggctcc aaggcgtaca
     4801 tcaagcaccc cgccgacatc cccgactacc tcaagcagtc cttccccgag ggcttcaagt
     4861 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcatccc
     4921 tgcaagacgg caccctcatc taccacgtca agttcatcgg cgtgaacttc ccctccgacg
     4981 gccccgtaat gcagaagaaa actcttggct gggagccctc cactgagcgc aactaccccc
     5041 gcgacggcgt gctgaagggc gagaaccaca tggcgctgaa gctgaagggc ggcggccact
     5101 acctgtgtga gttcaagtcc atctacatgg ctaagaagcc cgtgaagctg cccggctacc
     5161 actacgtgga ctacaagctc gacatcacct cccacaacga ggactacacc gtggtggagc
     5221 agtacgaacg cgccgaggcc cgccaccacc tgttccagta ggtttaaaca tcaacctctg
     5281 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5341 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5401 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5461 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5521 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5581 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5641 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     5701 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5761 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5821 cagacgagtc ggatctccct ttgggccgcc tccccgcctg gcctgcaggg gtacctttaa
     5881 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aagaggggac
     5941 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
     6001 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
     6061 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
     6121 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     6181 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
     6241 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
     6301 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
     6361 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
     6421 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     6481 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     6541 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     6601 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggccttg acattgctag
     6661 cgttttaccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     6721 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6781 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6841 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6901 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6961 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     7021 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     7081 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     7141 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7201 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     7261 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     7321 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     7381 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     7441 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7501 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7561 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7621 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7681 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7741 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7801 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7861 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7921 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7981 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     8041 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     8101 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     8161 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     8221 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     8281 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     8341 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     8401 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     8461 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8521 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8581 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8641 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8701 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8761 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8821 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcaact
     8881 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     8941 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     9001 atgtctggat caactggata actcaagcta accaaaatca tcccaaactt cccaccccat
     9061 accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg
     9121 aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac ttagtagtt
//
