LOCUS       sequence-430219-        8955 bp    DNA     circular SYN 15-APR-2025
DEFINITION  shRNAmir backbone for RNA interference, with GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8955)
  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 8955)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: LMAPGW
FEATURES             Location/Qualifiers
     source          1..8955
                     /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             join(4319..4321,4322..4324,4325..5038)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4364..4385)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4625..4644)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4972..4993
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5099..5119)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5517..5528)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5718..6351
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6480..6496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6480..6496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6493..6515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6504..6520
                     /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(6528..6534,6535..6552,6553..6558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6573..6594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6711..6728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6882..7470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6962..6981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7641..8432,8433..8501))
                     /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     8264..8283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8502..8606)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8654..8788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8691..8710)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8745..8764
                     /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 cccgctgagc
     4081 aatggaagcg ggtaggcctt gggggcagcg gccaatagca gctttgctcc ttcgctttcg
     4141 gggctcaggg gcggggaggg ggggggccgg ggggcgggct caggggcggg ctcaggggcg
     4201 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     4261 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctttaat taaccaccat
     4321 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     4381 cgacgtaaac ggccacaagt tcagcgtgtc tggcgagggc gagggcgatg ccacctacgg
     4441 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     4501 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     4561 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     4621 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     4681 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     4741 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     4801 catcaaggcg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     4861 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4921 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     4981 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagt
     5041 ttaaacatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5101 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5161 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5221 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5281 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5341 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5401 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     5461 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     5521 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     5581 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggcc
     5641 tgcaggggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     5701 aaaagaaaag aggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     5761 tctgtggatc taccacacac aaggctactt ccctgattag cagaactaca caccagggcc
     5821 aggggtcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagccaga
     5881 taaggtagaa gaggccaata aaggagagaa caccagcttg ttacaccctg tgagcctgca
     5941 tgggatggat gacccggaga gagaagtgtt agagtggagg tttgacagcc gcctagcatt
     6001 tcatcacgtg gcccgagagc tgcatccgga gtacttcaag aactgctgat atcgagcttg
     6061 ctacaaggga ctttccgctg gggactttcc agggaggcgt ggcctgggcg ggactgggga
     6121 gtggcgagcc ctcagatcct gcatataagc agctgctttt tgcctgtact gggtctctct
     6181 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     6241 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     6301 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     6361 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     6421 ggccttgaca ttgctagcgt tttaccgtcg acctctagct agagcttggc gtaatcatgg
     6481 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6541 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6601 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6661 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     7021 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     7081 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7141 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7201 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7261 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7321 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7381 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7441 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7501 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7561 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7621 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7681 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7741 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7801 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7861 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7921 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7981 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     8041 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     8101 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8161 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8221 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8281 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8341 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8401 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8461 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8521 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8581 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     8641 ggatcgggag atcaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     8701 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     8761 catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc aaaatcatcc
     8821 caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc atttactcta
     8881 aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt aaatatgtac
     8941 tacaaactta gtagt
//
