LOCUS       sequence-178323-        7515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miR30 based shMir knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Schwarzer A, Emmrich S, Schmidt F, Beck D, Ng M, Reimer C, Adams FF,
            Grasedieck S, Witte D, Kabler S, Wong JWH, Shah A, Huang Y, Jammal 
            R, Maroz A, Jongen-Lavrencic M, Schambach A, Kuchenbauer F, Pimanda 
            JE, Reinhardt D, Heckl D, Klusmann JH
  TITLE     The non-coding RNA landscape of human hematopoiesis and leukemia.
  JOURNAL   Nat Commun. 2017 Aug 9;8(1):218. doi: 10.1038/s41467-017-00212-4.
  PUBMED    28794406
REFERENCE   2  (bases 1 to 7515)
  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: "Nat 
            Commun."; date: "2017-08-9"; volume: "8(1):218. doi"; pages: " 
            10.1038/s41467-017-00212-4"
COMMENT     Sequence Label: pLKO5d.SFFV.eGFP-miR30n
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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    1753..1870
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1928..2335
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2341..2343,2344..2346,2347..3060)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2341..2343,2344..2346,2347..3057)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2386..2407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2647..2666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2994..3015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           3079..3200
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           3228..3354
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    3373..3961
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3426..3446)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3844..3855)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4033..4266
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4344..4465
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4381..4400)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4435..4454
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4505..4640
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4660..4679)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4661..4679)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4689..4706)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4689..4705)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4698..4720)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4847..5302
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4934..4953)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5144..5165
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5328..5432
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5433..5501,5502..6293)
                     /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     complement(5651..5670)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6464..7052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6953..6972
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7206..7223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7340..7361
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7376..7381,7382..7399,7400..7406)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7414..7430
                     /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)."
     primer_bind     7419..7441
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7438..7454
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7438..7454
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7473..7493
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7475..7493
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acaattctag atcttgagac aaatggcagt
     1741 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     1801 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     1861 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagcg
     1921 agctgcagta acgccatttt gcaaggcatg gaaaaatacc aaaccaagaa tagagaagtt
     1981 cagatcaagg gcgggtacat gaaaatagct aacgttgggc caaacaggat atctgcggtg
     2041 agcagtttcg gccccggccc ggggccaaga acagatggtc accgcagttt cggccccggc
     2101 ccgaggccaa gaacagatgg tccccagata tggcccaacc ctcagcagtt tcttaagacc
     2161 catcagatgt ttccaggctc ccccaaggac ctgaaatgac cctgcgcctt atttgaatta
     2221 accaatcagc ctgcttctcg cttctgttcg cgcgcttctg cttcccgagc tctataaaag
     2281 agctcacaac ccctcactcg gcgcgccagt cctccgacag actgagtcgg atccgccacc
     2341 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2401 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2461 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2521 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2581 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2641 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2701 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2761 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2821 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2881 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2941 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3001 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga
     3061 gcggccgcat attgctgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca
     3121 tcttggaaac acttgctggg attacttctt caggttaacc caacagaagg ctaaagaagg
     3181 tatattgctg ttgacagtga gcgggagacg tgattaccgt ctcttgccta ctgcctcgga
     3241 cttcaagggg ctactttagg agcaattatc ttgtttacta aaactgaata ccttgctatc
     3301 tctttgatac atttttacaa agctgaatta aaatggtata aattaaatca cttttttacg
     3361 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3421 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3481 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3541 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3601 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3661 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3721 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3781 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3841 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3901 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     3961 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4021 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     4081 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4141 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4201 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4261 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4321 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4381 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4441 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4501 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4561 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4621 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4681 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4741 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4801 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4861 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4921 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4981 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5041 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5101 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5161 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5221 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5281 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5341 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5401 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5461 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5521 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5581 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5641 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5701 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5761 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5821 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5881 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5941 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6001 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6061 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6121 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6181 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6241 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6301 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6361 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6421 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6481 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6541 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6601 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6661 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6721 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6781 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6841 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6901 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6961 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7021 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7081 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7141 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7201 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7261 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7321 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     7381 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7441 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
     7501 agctggagct gcaag
//
