LOCUS       sequence-494277-        4076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry plasmid with a ccdB cassette flanked with two inverted BsaI 
            restriction sites. Allows the high throughput cloning of cloning 
            inserts flanked with compatible 4-nt overhangs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4076)
  TITLE     Enhanced MicroRNA Accumulation and Gene Silencing Efficiency through
            Optimized Precursor Base Pairing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4076)
  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            "Unpublished"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: pENTR-BS-AtMIR390a-A18G-B/c
FEATURES             Location/Qualifiers
     source          1..4076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(730..735,736..753,754..760)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             814..1473
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(880..899)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1815..2120
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1999..2018
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2201..2300)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2317..2336)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2318..2336)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2341..2357)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2341..2357)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2459..2478)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2470..3279
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2537..2556)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3426..4014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3915..3934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gcggccgccc ccttcaccag tagagaagaa tctgtgagag accattaggc
      721 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggag
      781 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
      841 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
      901 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
      961 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1021 catccggagt tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1081 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1141 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1201 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1261 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1321 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1381 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1441 cagtactgcg atgagtggca gggcggggcg taaacgcgtg gagccggctt actaaaagcc
     1501 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     1561 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg
     1621 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     1681 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     1741 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     1801 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     1861 tgtttgtgga tgtacagagt gatattattg acacgcccgg ccgacggatg gtgatccccc
     1921 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     1981 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtttccgtta
     2041 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     2101 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcacctcgac
     2161 ggtctcacat tggctcttct tactaagggt gggcgcgccg acccagcttt cttgtacaaa
     2221 gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt
     2281 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     2341 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     2401 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     2461 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     2521 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2581 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2641 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2701 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2761 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2821 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2881 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2941 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     3001 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     3061 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     3121 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     3181 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     3241 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     3301 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     3361 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3421 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3481 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3541 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3601 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3661 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3721 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3781 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3841 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3901 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3961 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4021 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
