LOCUS       sequence-430336-        8006 bp    DNA     circular SYN 15-DEC-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 8006)
  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 8006)
  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: MAPGW
FEATURES             Location/Qualifiers
     source          1..8006
                     /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"
     CDS             join(3011..3013,3014..3016,3017..3730)
                     /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(3056..3077)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3317..3336)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3664..3685
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3791..3811)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4209..4220)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4401..4915
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5077..5093
                     /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(5101..5107,5108..5125,5126..5131))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5146..5167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5284..5301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5455..6043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5535..5554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6214..7005,7006..7074))
                     /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     6837..6856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7075..7179)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7247..7265
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7303..7325)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7425..7444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7638..7660
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7788..7807
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3061 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga
     3121 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3181 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     3241 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3301 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3361 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3421 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3481 gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3541 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3601 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3661 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3721 gtacaagtaa gtttaaacat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3781 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3841 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3901 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3961 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     4021 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     4081 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     4141 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     4201 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4261 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4321 ccccgcctgg gtcgacctgc aggcatgcaa gcttatcgat aaaataaaag attttattta
     4381 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     4441 taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag
     4501 gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc
     4561 tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt
     4621 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     4681 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     4741 caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc
     4801 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     4861 tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatgggta
     4921 acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc
     4981 tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa tagttcatta
     5041 tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca caattccaca
     5101 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5881 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7201 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     7261 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     7321 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     7381 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     7441 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     7501 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     7561 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     7621 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca
     7681 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     7741 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     7801 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     7861 tagaggcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg
     7921 actcaacaat atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt
     7981 ttatttagtc tccagaaaaa gggggg
//
