LOCUS       Exported                8008 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  miR-E (miR-30 variant)-based RNAi with GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    LENG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8008)
  AUTHORS   Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M, Roth M, 
            Lai DY, Barbosa IA, Kwon JS, Guan Y, Sinha N, Zuber J
  TITLE     An optimized microRNA backbone for effective single-copy RNAi.
  JOURNAL   Cell Rep. 2013 Dec 26;5(6):1704-13. doi: 
            10.1016/j.celrep.2013.11.020. Epub 2013 Dec 12.
  PUBMED    24332856
REFERENCE   2  (bases 1 to 8008)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8008
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             101..617
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    681..1022
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1089..1505
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1435..1457
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1473..1489
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1779..2278
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1814..1837)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2211..2230
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2292..3095
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2355..2374)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2965..2984
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3118..3670
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3262..3279)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3489..3508
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3682..4401
                     /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(3727..3748)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3988..4007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4335..4356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4503..5017
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5179..5195
                     /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(5203..5233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5248..5269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5386..5403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5557..6145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5637..5656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6316..7176)
                     /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     6939..6958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7177..7281)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7349..7367
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7405..7427)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7527..7546
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7740..7762
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7890..7909
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga
       61 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
      121 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag
      181 aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg
      241 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
      301 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
      361 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
      421 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
      481 cctccgatag actgcgtcgc ccgggtaccc gtattcccaa taaagcctct tgctgtttgc
      541 atccgaatcg tggactcgct gatccttggg agggtctcct cagattgatt gactgcccac
      601 ctcgggggtc tttcatttgg aggttccacc gagatttgga gacccctgcc cagggaccac
      661 cgaccccccc gccgggaggt aagctggcca gcggtcgttt cgtgtctgtc tctgtctttg
      721 tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct gtactagtta gctaactagc
      781 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
      841 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
      901 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
      961 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     1021 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     1081 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     1141 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     1201 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     1261 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     1321 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     1381 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     1441 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     1501 gcgccggaat tagatctctc gactagggat aacagggtaa ttgtttgaat gaggcttcag
     1561 tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta cttcgacttc
     1621 ttaacccaac agaaggctcg agcaaccaga attcgagcaa ttatcttgtt tactaaaact
     1681 gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg gtataaatta
     1741 aatcactttt ttcaattgac gcgtgatcta attctaccgg gtaggggagg cgcttttccc
     1801 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     1861 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     1921 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     1981 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     2041 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     2101 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     2161 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     2221 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     2281 ctgcagccaa tatgggatcg gccattgaac aagatggatt gcacgcaggt tctccggccg
     2341 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     2401 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     2461 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     2521 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     2581 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     2641 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     2701 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     2761 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     2821 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     2881 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     2941 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3001 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     3061 tcgccttcta tcgccttctt gacgagttct tctgagcaat tccgcccccc ccccctaacg
     3121 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
     3181 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
     3241 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
     3301 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
     3361 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
     3421 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
     3481 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
     3541 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     3601 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
     3661 gatgataata tggccacaac catggtgagc aagggcgagg agctgttcac cggggtggtg
     3721 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtctggcgag
     3781 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     3841 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     3901 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3961 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     4021 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4081 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     4141 atggccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatcgag
     4201 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     4261 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     4321 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     4381 atggacgagc tgtacaagta atgaattaat taagaattat caagcttatc gataccgtcg
     4441 acctgcagcc aagcttatcg ataaaataaa agattttatt tagtctccag aaaaaggggg
     4501 gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
     4561 catggaaaat acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca
     4621 gcagaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     4681 agaacagatg gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg
     4741 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     4801 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     4861 cccctcactc ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca
     4921 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     4981 ctgagtgatt gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg
     5041 agaacaacat tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg
     5101 ttttgaatcc acatactcca atactcctga aatagttcat tatggacagc gcagaaagag
     5161 ctggggagaa ttgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5221 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5281 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5341 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5401 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5461 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5521 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5581 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5641 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5701 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5761 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5821 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5881 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5941 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6001 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6061 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6121 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6181 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6241 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6301 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6361 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6421 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6481 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6541 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6601 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6661 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6721 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6781 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6841 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6901 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6961 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7021 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7081 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7141 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7201 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7261 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     7321 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     7381 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     7441 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     7501 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     7561 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
     7621 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     7681 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     7741 ccagtcacga cgttgtaaaa cgacggcgca aggaatggtg catgcaagga gatggcgccc
     7801 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     7861 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     7921 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggcg attagtccaa
     7981 tttgttaaag acaggatatc agtggtcc
//
