LOCUS       sequence-409378-        8003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNAmir backbone for RNA interference, with Azurite reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8003)
  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 8003)
  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: MAPZW
FEATURES             Location/Qualifiers
     source          1..8003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2494..2993
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2529..2552)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2926..2945
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(3314..3333)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3788..3808)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4206..4217)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4398..4912
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5074..5090
                     /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(5098..5104,5105..5122,5123..5128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5143..5164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5281..5298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5452..6040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5532..5551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6211..7002,7003..7071))
                     /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     6834..6853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7072..7176)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7244..7262
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7300..7322)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7422..7441
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7635..7657
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7785..7804
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatctctc gactagggat aacagggtaa
     1441 ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt
     1501 gctgggatta cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga
     1561 caggatccac attaacaact ttaagtccag caatttgagt taagggtgtt gctctcaatg
     1621 atttcactaa tggttcaatt tttaatttct tttcttctgg ttcaaaattc aagtttcaag
     1681 tgaaagtgta atatgcaccc atttctttca ataaatcttc taaatagtcc actaatgttt
     1741 tattttgttt tttacaaaat caagcagcct ctgctatcaa tatagaacct tgtattccat
     1801 ctttatctct agctgagtca tcaattacat atccataact ttcttcataa gcaaaaacaa
     1861 aatttaatcc gttatcttct tctttagcaa tttctctacc cattcatttc aatccagtta
     1921 aagtttttac aatcttaact ccatattttt cctgagcgat tctatcaccc aaatcacttg
     1981 ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt tttagatttg
     2041 ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt acaaaatgac
     2101 catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata ataataatat
     2161 ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca aattctggat
     2221 ttggatttac aacatttttc aatgtttcat cttcaaatgc acgatcttca ccctcaataa
     2281 cgttatatcc tgattcacgt aatctttttg gggtaaattt agttcctgtt ccattaactg
     2341 cgctaaaaat agggccctgc ctcggacttc aaggggctag aattcgagca attatcttgt
     2401 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     2461 ggtataaatt aaatcacttt tttcaattct accgggtagg ggaggcgctt ttcccaaggc
     2521 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     2581 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     2641 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     2701 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     2761 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     2821 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     2881 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     2941 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgaccttta
     3001 attaaccacc atgagcaaag gcgaagagct gttcaccggc gtggtgccca tcctggtgga
     3061 gctggacggc gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc
     3121 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3181 gcccaccctg gtgaccaccc tgagccacgg cgtgcagtgc ttcagccggt accccgacca
     3241 catgaagcag cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagcggac
     3301 catcttcttc aaggacgacg gcaactacaa gacccgggcc gaggtgaagt tcgagggcga
     3361 caccctggtg aaccggatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3421 gggccacaag ctggagtaca acttcaacag ccacaacatc tacatcatgg ccgacaagca
     3481 gaagaacggc atcaaggtga acttcaagat ccggcacaac atcgaggacg gcagcgtgca
     3541 gctggccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3601 caaccactac ctgagcaccc agagcgccct gagcaaggac cccaacgaga agcgggacca
     3661 catggtgctg ctggagttcc ggaccgccgc cggcatcacc cacggcatgg acgagctcta
     3721 taagtaagtt taaacatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3781 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3841 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3901 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3961 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4021 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     4081 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     4141 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     4201 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     4261 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4321 cgcctgggtc gacctgcagg catgcaagct tatcgataaa ataaaagatt ttatttagtc
     4381 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     4441 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt
     4501 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     4561 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta
     4621 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     4681 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     4741 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     4801 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     4861 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca
     4921 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga
     4981 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg
     5041 acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa ttccacacaa
     5101 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5221 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5281 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5341 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5401 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5461 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5521 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5581 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5641 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5701 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5761 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5821 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5881 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5941 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6001 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6061 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6121 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6181 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6241 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6301 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6361 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6421 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6481 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6541 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6601 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6661 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6721 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6781 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6841 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6901 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6961 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7021 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7081 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7141 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7201 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     7261 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     7321 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     7381 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     7441 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     7501 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7561 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7621 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc
     7681 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7741 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7801 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7861 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact
     7921 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     7981 tttagtctcc agaaaaaggg ggg
//
