LOCUS       sequence-409118-        9030 bp    DNA     circular SYN 15-APR-2025
DEFINITION  shRNAmir backbone for RNA interference, with Ametrine reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9030)
  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 9030)
  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: LCAPAW
FEATURES             Location/Qualifiers
     source          1..9030
                     /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)"
     enhancer        2201..2504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2505..2708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2659..2679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2705..2729
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3877..4376
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3912..3935)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4309..4328
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(4394..4396,4397..4399,4400..5113)
                     /codon_start=1
                     /product="photostable yellow fluorescent protein with a 
                     large Stokes shift, excitable with violet light (Ai et al.,
                     2008)"
                     /label=mAmetrine1.1
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVRGEGEGDATNGKLTL
                     KFICTSGELPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNAWDAYITADKQKNGIK
                     ANFKIEHNVKDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(4439..4460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5174..5194)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5592..5603)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5793..6426
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6555..6571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6555..6571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6568..6590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6579..6595
                     /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(6603..6609,6610..6627,6628..6633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6648..6669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6786..6803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6957..7545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7037..7056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7716..8507,8508..8576))
                     /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     8339..8358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8577..8681)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8729..8863
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8766..8785)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8820..8839
                     /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 cgataagctt gggagttccg cgttacataa cttacggtaa
     2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     2761 tccatagaag acaccgactc tactagagga tcgctagcgc ttcgactagg gataacaggg
     2821 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     2881 cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt atattgctgt
     2941 tgacaggatc cacattaaca actttaagtc cagcaatttg agttaagggt gttgctctca
     3001 atgatttcac taatggttca atttttaatt tcttttcttc tggttcaaaa ttcaagtttc
     3061 aagtgaaagt gtaatatgca cccatttctt tcaataaatc ttctaaatag tccactaatg
     3121 ttttattttg ttttttacaa aatcaagcag cctctgctat caatatagaa ccttgtattc
     3181 catctttatc tctagctgag tcatcaatta catatccata actttcttca taagcaaaaa
     3241 caaaatttaa tccgttatct tcttctttag caatttctct acccattcat ttcaatccag
     3301 ttaaagtttt tacaatctta actccatatt tttcctgagc gattctatca cccaaatcac
     3361 ttgttacaaa acttgaatat agagccggat tttttggaat gctatttaag cgttttagat
     3421 ttgataattt tcaatcaatt aaaattggtc ctgtttgatt tccatctaat cttacaaaat
     3481 gaccatcatg ttttattgcc attccaaatc tgtcagcatc tgggtcattc ataataataa
     3541 tatctgcatc atgtttaata ccatattcaa gcggtatttt tcatgcagga tcaaattctg
     3601 gatttggatt tacaacattt ttcaatgttt catcttcaaa tgcacgatct tcaccctcaa
     3661 taacgttata tcctgattca cgtaatcttt ttggggtaaa tttagttcct gttccattaa
     3721 ctgcgctaaa aatagggccc tgcctcggac ttcaaggggc tagaattcga gcaattatct
     3781 tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa
     3841 aatggtataa attaaatcac ttttttcaat tctaccgggt aggggaggcg cttttcccaa
     3901 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     3961 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     4021 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     4081 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     4141 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     4201 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     4261 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     4321 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     4381 ttaattaacc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4441 ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgcgcggcg agggcgaggg
     4501 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc acctccggcg agctgcccgt
     4561 gccctggccc accctcgtga ccaccctgtc ttacggcgtg cagtgcttcg cccgctaccc
     4621 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     4681 gcgcaccatc tccttcaagg acgacggcag ctacaggacc cgcgccgagg tgaagttcga
     4741 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     4801 catcctgggg cacaagctgg agtacaacat gaacgcttgg gacgcttata tcacggccga
     4861 caagcagaag aacggcatca aagcgaactt caagatcgag cacaacgtca aggacggcgg
     4921 cgtgcagctc gccgacgctt accagcagaa cacccccatc ggcgacggct ccgtgctgct
     4981 gcctgacaac cactacctga gcttccagag caagctgttc aaagacccca acgagcagcg
     5041 cgatcacatg gtcctgctgg agttcgttac cgccgccggg ataactcccg gcatggacga
     5101 gctgtacaag taagtttaaa catcaacctc tggattacaa aatttgtgaa agattgactg
     5161 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     5221 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     5281 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     5341 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     5401 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     5461 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     5521 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     5581 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     5641 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5701 cctccccgcc tggcctgcag gggtaccttt aagaccaatg acttacaagg cagctgtaga
     5761 tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact cccaacgaag
     5821 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attagcagaa
     5881 ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt
     5941 accagttgag ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca
     6001 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt ggaggtttga
     6061 cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg
     6121 ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga ggcgtggcct
     6181 gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg ctttttgcct
     6241 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     6301 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6361 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6421 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     6481 tatcagagag tgagaggcct tgacattgct agcgttttac cgtcgacctc tagctagagc
     6541 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6601 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6661 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6721 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     6781 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6841 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6901 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6961 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     7021 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     7081 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     7141 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     7201 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     7261 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     7321 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     7381 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     7441 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     7501 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     7561 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     7621 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7681 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7741 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7801 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7861 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7921 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7981 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     8041 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     8101 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     8161 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     8221 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     8281 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     8341 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     8401 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     8461 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     8521 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     8581 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     8641 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     8701 ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata atggttacaa
     8761 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     8821 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc
     8881 taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg
     8941 gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat
     9001 ttgttaaata tgtactacaa acttagtagt
//
