LOCUS       sequence-202521-        3920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for the overexpression of miR-301a 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 3920)
  TITLE     Mannironi Lab miR constructs
REFERENCE   2  (bases 1 to 3920)
  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: SP65U1-301a
FEATURES             Location/Qualifiers
     source          1..3920
                     /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(1005..1023)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1006..1023)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1313..1332)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1546..1565)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1665..1687
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1725..1743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1811..1915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1916..1984,1985..2776)
                     /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(2134..2153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2947..3535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3436..3455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3689..3706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3823..3844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3859..3864,3865..3882,3883..3889)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3897..3913
                     /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(3902..3920,1..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 gatctgcatg tttatgtctg aatgctgcta
      481 atttcaggtt agtcaacaaa aaccaattat cactttggtc ctttgccctt ccaaaaatat
      541 actggtataa atgtttttag gttttggatg tcttctgact aaaatggcaa ctcaacttta
      601 aggtctttgc acactgagcc ttgcattcct ttcctgctca ctcctgctaa cggctgctct
      661 gactttattg cactactgta ctttacagcg agcagtgcaa tagtattgtc aaagcatccg
      721 cgagcaggtt gcacaccttt cctggttctt gtcttacatc ttcagtattt gtgaaaaggt
      781 ttctataact ttcagtgtgg aaaactgtat aattttgtta ctatgtatat tttttagaaa
      841 gtcatcattt gaaaaaatat cattaaatta acatttatca tttctgtatg gcactgaaaa
      901 ttcattactg tacgcggtac caggcctctc gagctgactt tctggagttt caaaagtaga
      961 ctgtacgcta accggatccc cgggcgagct cgaattccgt gtattctata gtgtcaccta
     1021 aatcgtatgt gtatgataca taaggttatg tattaattgt agccgcgttc taacgacaat
     1081 atgtacaagc ctaattgtgt agcatctggc ttactgaagc agaccctatc atctctctcg
     1141 taaactgccg tcagagtcgg tttggttgga cgaaccttct gagtttctgg taacgccgtc
     1201 ccgcacccgg aaatggtcag cgaaccaatc agcagggtca tcgctagcca gatcctctac
     1261 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     1321 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     1381 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     1441 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     1501 atgcaggagt cgcataaggg agagcgtcga atggtgcact ctcagtacaa tctgctctga
     1561 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     1621 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     1681 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     1741 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1801 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1861 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1921 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1981 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2041 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2101 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2161 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2221 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2281 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2341 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2401 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2461 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2521 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2581 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2641 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2701 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2761 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2821 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2881 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2941 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3001 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3061 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3121 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3181 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3241 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3301 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3361 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3421 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3481 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3541 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3601 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3661 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3721 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3781 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3841 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3901 gagcggataa caatttcaca
//
