LOCUS       sequence-222779-        5771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miR-30a RNAi Expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5771)
  TITLE     Mmu-miR-30a Marc Beltra plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5771)
  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: 
            "Unpublished"
COMMENT     Sequence Label: mmu-miR-30a natural design (NAT)
FEATURES             Location/Qualifiers
     source          1..5771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    750..774
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(783..785,786..788,789..1502)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(828..849)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1089..1108)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1436..1457
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1562..1588
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1664..1705
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(1734..1758)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1800..1819)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1844..1892
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        complement(2063..2081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2086..2102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2086..2102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      2170..2598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2257..2276)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2467..2488
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2607..2627)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2612..2941
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2792..2927
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2854..2873
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2989..3036
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3055..3453
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3611..3732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3648..3667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3702..3721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3767..3784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3938..4526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4018..4037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4622..5413)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagtcccaag ctggctagtt aagctatcaa caagtttgta caaaaaagca ggctttaaaa
      781 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      841 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      901 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      961 ccctcgtgac caccttcacc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga
     1021 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1081 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1141 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1201 acaagctgga gtacaactac aacagccaca aggtctatat caccgccgac aagcagaaga
     1261 acggcatcaa ggtgaacttc aagacccgcc acaacatcga ggacggcagc gtgcagctcg
     1321 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1381 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1441 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1501 aagctaagca cttcgtggcc gtcgatcgtt taaagggagg tagtgagtcg accagtggat
     1561 cctggaggct tgctgaaggc tgtatgctgg cgactgtaaa catcctcgac tggaagctgt
     1621 gaagccacaa atgggctttc agtcggatgt ttgcagctgc caggacacaa ggcctgttac
     1681 tagcactcac atggaacaaa tggcccagat ctggccgcac tcgagatatc tagacccagc
     1741 tttcttgtac aaagtggttg atctagaggg cccgcggttc gctgatgggg gaggctaact
     1801 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     1861 aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     1921 gcactctgtc gataccccac cgtgacccca ttggggccaa tacgcccgcg tttcttcctt
     1981 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     2041 ggcaggccct gccatagcat cccctatagt gagtcgtatt acatggtcat agctgtttcc
     2101 tggcagctct ggcccgtgtc tcaaaatctc tgatggatct gcgcagctgg ggctctaggg
     2161 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2221 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2281 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2341 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2401 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2461 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2521 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2581 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2641 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2701 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2761 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2821 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2881 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2941 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     3001 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     3061 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     3121 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     3181 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     3241 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     3301 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     3361 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     3421 ttgctgccct ctggttatgt gtgggagggc taagcacttc gtggccgagg agcaggactg
     3481 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3541 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3601 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3661 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3721 caatgtatct tatcatgtct gtataccgtc gctcttccgc tgcttcctcg ctcactgact
     3781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4561 ctcagtggaa cgacgcgtaa ctcacgttaa gggattttgg tcatgggtgg ctcgacgagg
     4621 gttatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat tcttccaact
     4681 gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt ctagcttcaa
     4741 gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca gcgacatcct
     4801 tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta agcactacat
     4861 ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt tcatttagcg
     4921 cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct ggacctacca
     4981 aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg tcgatcgtgg
     5041 ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat tgcagttcgc
     5101 gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg acttctacag
     5161 cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag
     5221 ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca atatcactgt
     5281 gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc aacgtcggtt
     5341 cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact tcggcgatca
     5401 ccgcttccct cataatgttt aactttgttt tagggcgact gccctgctgc gtaacatcgt
     5461 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc
     5521 gaggcataga ctgtacccca aaaaaacagt cataacaagc catgaaaacc gccactgcgc
     5581 cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc atacgctact
     5641 tgcattacag cttacgaacc gaacaggctt atgtccactg ggttcgtgcc ttcatccgtt
     5701 tccacggtgt gcgtcacccg gcaaccttgg gtagcagcga agtcgaggca tttctgtcct
     5761 ggctggtcta g
//
