LOCUS       sequence-178322-        7500 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 7500)
  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 7500)
  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.dTomato-miR30n
FEATURES             Location/Qualifiers
     source          1..7500
                     /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             2341..3042
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(2474..2492)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2742..2761
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2936..2959
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           3064..3185
                     /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           3213..3339
                     /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    3358..3946
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3411..3431)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3829..3840)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4018..4251
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4329..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4366..4385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4420..4439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4490..4625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4645..4664)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4646..4664)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4674..4691)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4674..4690)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4683..4705)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4832..5287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4919..4938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5129..5150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5313..5417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5418..5486,5487..6278)
                     /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(5636..5655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6449..7037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6938..6957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7191..7208
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7325..7346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7361..7366,7367..7384,7385..7391)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7399..7415
                     /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     7404..7426
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7423..7439
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7423..7439
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7458..7478
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7460..7478
                     /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 ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag
     2401 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     2461 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
     2521 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc
     2581 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2641 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc
     2701 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag
     2761 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
     2821 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc
     2881 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg
     2941 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc
     3001 cgccaccacc tgttcctgta cggcatggac gagctgtaca agtgagcggc cgcatattgc
     3061 tgtttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg gaaacacttg
     3121 ctgggattac ttcttcaggt taacccaaca gaaggctaaa gaaggtatat tgctgttgac
     3181 agtgagcggg agacgtgatt accgtctctt gcctactgcc tcggacttca aggggctact
     3241 ttaggagcaa ttatcttgtt tactaaaact gaataccttg ctatctcttt gatacatttt
     3301 tacaaagctg aattaaaatg gtataaatta aatcactttt ttacgcgtta agtcgacaat
     3361 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3421 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3481 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3541 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3601 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3661 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3721 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3781 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3841 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3901 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     3961 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     4021 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     4081 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     4141 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     4201 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     4261 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     4321 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4381 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4441 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     4501 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4561 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4621 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     4681 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4741 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4801 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4861 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4921 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4981 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5041 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5101 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5161 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5221 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5281 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5341 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5401 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5461 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5521 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5581 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5641 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5701 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5761 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5821 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5881 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5941 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6001 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6061 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6121 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6181 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6241 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6301 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6361 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6421 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6481 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6541 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6601 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6661 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6721 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6781 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6841 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6901 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6961 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7021 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7081 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7141 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7201 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7261 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7321 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     7381 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     7441 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
//
