LOCUS       sequence-202522-        3809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for the overexpression of miR-135a under the U1 expression 
            cassette inserted in the pSP65 plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3809)
  TITLE     Mannironi Lab miR constructs
REFERENCE   2  (bases 1 to 3809)
  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"
COMMENT     Sequence Label: SP65U1-135a-2
FEATURES             Location/Qualifiers
     source          1..3809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(894..912)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(895..912)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1202..1221)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1435..1454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1554..1576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1614..1632)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1700..1804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1805..1873,1874..2665)
                     /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(2023..2042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2836..3424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3325..3344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3578..3595
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3712..3733
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3748..3753,3754..3771,3772..3778)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3786..3802
                     /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     join(3791..3809,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcttgggct gcaggtcgac tctagaggat
       61 ccggtaagga ccagcttctt tgggagagaa cagacgcagg ggcgggaggg aaaaagggag
      121 aggcagacgt cacttcccct tggcggctct ggcagcagat tggtcggttg agtggcagaa
      181 aggcagacgg ggactgggca aggcactgtc ggtgacatca cggacagggc gacttctatg
      241 tagatgaggc agcgcagagg ctgctgcttc gccacttgct gcttcaccac gaaggagttc
      301 ccgtgccctg ggagcgggtt caggaccgct gatcggaagt gagaatccca gctgtgtgtc
      361 agggctggaa agggctcggg agtgcgcggg gcaagtgacc gtgtgtgtaa agagtgaggc
      421 gtatgaggct gtgtcggggc agaggcccaa gatctatcta agtgtgcttt gtgtccctta
      481 catgtagcct aaaatgttcc ttaatgtagc atgaaatgat tggttagctt ggaaatggtt
      541 ttgaagtcgt gtgaagaaaa taagttttac atccgaccaa gataaattca ctctagtgct
      601 ttatggcttt ttattcctat gtgatcgtaa taaagtctca tgtagggatg gaagccatga
      661 aatacattgt gaaaattcat caactaagaa ggggccatca ggatagcaaa ctatagcctg
      721 tggaccgtgc tggagacgtg agctagtgga catctctgct tccctctttg gtgttccttg
      781 agtgtcttta tccttaggca ttgaggtacc aggcctctcg agctgacttt ctggagtttc
      841 aaaagtagac tgtacgctaa ccggatcccc gggcgagctc gaattccgtg tattctatag
      901 tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct
      961 aacgacaata tgtacaagcc taattgtgta gcatctggct tactgaagca gaccctatca
     1021 tctctctcgt aaactgccgt cagagtcggt ttggttggac gaaccttctg agtttctggt
     1081 aacgccgtcc cgcacccgga aatggtcagc gaaccaatca gcagggtcat cgctagccag
     1141 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     1201 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     1261 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     1321 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     1381 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc tcagtacaat
     1441 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     1501 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     1561 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     1621 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1681 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1741 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1801 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1861 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1921 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1981 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2041 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2101 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2161 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2221 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2281 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2341 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2401 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2461 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2521 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2581 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2641 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2701 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2761 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2821 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2881 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2941 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3001 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3061 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3121 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3181 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3241 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3301 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3361 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3421 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3481 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3541 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3601 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     3661 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     3721 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     3781 tggaattgtg agcggataac aatttcaca
//
