LOCUS       sequence-410144-        8889 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 8889)
  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 8889)
  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: LHAPOW
FEATURES             Location/Qualifiers
     source          1..8889
                     /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..2396
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        3745..4244
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3780..3803)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4177..4196
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(5033..5053)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5451..5462)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5652..6285
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6414..6430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6414..6430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6427..6449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6438..6454
                     /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(6462..6468,6469..6486,6487..6492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6507..6528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6645..6662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6816..7404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6896..6915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7575..8366,8367..8435))
                     /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     8198..8217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8436..8540)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8588..8722
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8625..8644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8679..8698
                     /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 cgatgggcag agcgcacatc gcccacagtc cccgagaagt
     2221 tggggggagg ggtcggcaat tgaaccggag cctagagaag gtggcgcggg gtaaactggg
     2281 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2341 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagggct
     2401 cgcatctctc cttcacgcgc ccgccgcctt acctgaggcc gccatccacg ccggttgagt
     2461 cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaacta cgtccgccgt ctaggtaagt
     2521 ttagagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc
     2581 agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt
     2641 tctgttctgc gccgttacag atcagcgctt cgactaggga taacagggta attgtttgaa
     2701 tgaggcttca gtactttaca gaatcgttgc ctgcacatct tggaaacact tgctgggatt
     2761 acttcgactt cttaacccaa cagaaggctc gagaaggtat attgctgttg acaggatcca
     2821 cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat gatttcacta
     2881 atggttcaat ttttaatttc ttttcttctg gttcaaaatt caagtttcaa gtgaaagtgt
     2941 aatatgcacc catttctttc aataaatctt ctaaatagtc cactaatgtt ttattttgtt
     3001 ttttacaaaa tcaagcagcc tctgctatca atatagaacc ttgtattcca tctttatctc
     3061 tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca aaatttaatc
     3121 cgttatcttc ttctttagca atttctctac ccattcattt caatccagtt aaagttttta
     3181 caatcttaac tccatatttt tcctgagcga ttctatcacc caaatcactt gttacaaaac
     3241 ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt gataattttc
     3301 aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga ccatcatgtt
     3361 ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata tctgcatcat
     3421 gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga tttggattta
     3481 caacattttt caatgtttca tcttcaaatg cacgatcttc accctcaata acgttatatc
     3541 ctgattcacg taatcttttt ggggtaaatt tagttcctgt tccattaact gcgctaaaaa
     3601 tagggccctg cctcggactt caaggggcta gaattcgagc aattatcttg tttactaaaa
     3661 ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa tggtataaat
     3721 taaatcactt ttttcaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3781 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3841 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3901 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3961 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     4021 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     4081 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     4141 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     4201 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgaccttt aattaaccac
     4261 catggtgagc aagggcgaag agaataacat ggcaatcatc aaggagttca tgcgcttcaa
     4321 ggttcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     4381 ccgcccctac gagggctttc agaccgctaa gctgaaggtg accaagggtg gccccctgcc
     4441 cttcgcctgg gacatcctgt cccctcagtt cacctacggc tccaaggcat acgtgaagca
     4501 ccccgccgac atccccgact acttcaagct gtccttcccc gagggcttca agtgggagcg
     4561 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcgt ccctgcaaga
     4621 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     4681 aatgcagaaa aagactatgg gctgggaggc atcctccgag cggatgtacc ccgaggacgg
     4741 cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacacctc
     4801 cgaggtcaag accacctaca aggccaagaa gcccgtgcaa ctgcccggcg cctacatcgt
     4861 cggcatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     4921 acgcgccgag ggccgccact ccaccggcgg catggacgag ctatacaagt aggtttaaac
     4981 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     5041 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     5101 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     5161 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     5221 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     5281 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5341 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg
     5401 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5461 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5521 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct ggcctgcagg
     5581 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     5641 aaagagggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     5701 gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt
     5761 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt
     5821 agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat
     5881 ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca
     5941 cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa
     6001 gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg
     6061 agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag
     6121 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6181 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     6241 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     6301 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt
     6361 gacattgcta gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     6421 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     6481 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6541 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6601 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6661 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6721 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6781 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6841 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6901 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6961 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7021 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7081 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7141 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7201 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7261 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7321 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7381 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7441 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7501 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7561 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7621 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7681 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7741 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7801 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7861 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7921 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7981 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     8041 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     8101 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     8161 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8221 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8281 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8341 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8401 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8461 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8521 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     8581 ggagatcaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     8641 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     8701 tgtatcttat catgtctgga tcaactggat aactcaagct aaccaaaatc atcccaaact
     8761 tcccacccca taccctatta ccactgccaa ttacctgtgg tttcatttac tctaaacctg
     8821 tgattcctct gaattatttt cattttaaag aaattgtatt tgttaaatat gtactacaaa
     8881 cttagtagt
//
