LOCUS       sequence-12075-e        5309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning miR30-based shRNA driven by TRE promoter 
            with dsRed2 coexpression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5309)
  AUTHORS   Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang JI, 
            Rebres R, Roach T, Seaman W, Simon MI, Fraser ID
  TITLE     A single lentiviral vector platform for microRNA-based conditional 
            RNA interference and coordinated transgene expression.
  JOURNAL   Proc Natl Acad Sci U S A. 2006 Sep 12. 103(37):13759-64.
  PUBMED    16945906
REFERENCE   2  (bases 1 to 5309)
  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: "Proc Natl 
            Acad Sci U S A. 2006 Sep 12. 103(37):13759-64."
COMMENT     Sequence Label: pEN_TRmiRc2
FEATURES             Location/Qualifiers
     source          1..5309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..48
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    67..166
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    join(205..223,224..246,247..265,266..288,289..307,308..330,
                     331..349,350..372,373..391,392..414,415..433,434..456,
                     457..475)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        508..546
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..567
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             653..1327
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(833..853)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1245..1268
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    1328..1384
                     /label=MCS
                     /note="multiple cloning site"
     ncRNA           1431..1552
                     /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"
     CDS             1588..1893
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1772..1791
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     ncRNA           1914..2040
                     /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"
     protein_bind    complement(2079..2178)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2290..2309)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2368..2387)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2961..3494)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3683..3688,3689..3705,3706..3711))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4368..4956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4857..4876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5121..5207
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(5299..5309,1..17)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc
       61 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
      121 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctttaa aggaaccaat
      181 tcagtcgact ggatccttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      241 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      301 atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt
      361 cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta
      421 tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt
      481 gaaagtcgag ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc
      541 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
      601 catagaagac accgggaccg atccagcctc cgcggccccg aactagtcca ccatggcctc
      661 ctccgagaac gtcatcaccg agttcatgcg cttcaaggtg cgcatggagg gcaccgtgaa
      721 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gccacaacac
      781 cgtgaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc
      841 ccagttccag tacggctcca aggtgtacgt gaagcacccc gccgacatcc ccgactacaa
      901 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
      961 cgtggcgacc gtgacccagg actcctccct gcaggacggc tgcttcatct acaaggtgaa
     1021 gttcatcggc gtgaacttcc cctccgacgg ccccgtgatg cagaagaaga ccatgggctg
     1081 ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg agacccacaa
     1141 ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagtcca tctacatggc
     1201 caagaagccc gtgcagctgc ccggctacta ctacgtggac gccaagctgg acatcacctc
     1261 ccacaacgag gactacacca tcgtggagca gtacgagcgc accgagggcc gccaccacct
     1321 gttcctgaga tctcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg
     1381 atccaccgga tctagataaa ctagtcgact agggataaca gggtaattgt ttgaatgagg
     1441 cttcagtact ttacagaatc gttgcctgca catcttggaa acacttgctg ggattacttc
     1501 ttcaggttaa cccaacagaa ggctcgagaa ggtatattgc tgttgacagt gagcgtgctg
     1561 caggtaagct tgaggtgtgc ttccagaatg cagtttaagg tttacaccta taaaagagag
     1621 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg
     1681 atagtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac
     1741 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg
     1801 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa
     1861 aacgccatta acctgatgtt ctggggaata tagaattcca acctgctgct tgcctactgc
     1921 ctcggaattc aaggggctac tttaggagca attatcttgt ttactaaaac tgaatacctt
     1981 gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaatt aaatcacttt
     2041 tttcaattgg aagactaatg cgttttcgag atatctagac ccagctttct tgtacaaagt
     2101 tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     2161 aaataaaatc attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat
     2221 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     2281 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     2341 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     2401 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     2461 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     2521 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     2581 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     2641 caggtgaaaa tattgttgat gcgctggcag tgtccctgcg ccggttgcat tcgattcctg
     2701 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     2761 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     2821 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     2881 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaggc cgggaagccg
     2941 atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact
     3001 tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
     3061 tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
     3121 gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc
     3181 tcactacgcg gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc aacaaccgct
     3241 tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa
     3301 tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
     3361 tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg
     3421 cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg
     3481 ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
     3541 ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac aagccatgaa
     3601 aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga
     3661 gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg
     3721 tgccttcatc cgtttccacg gtgtgcgtca cccggcaacc ttgggcagca gcgaagtcga
     3781 ggcatttctg tcctggctgg cgaacgagcg caaggtttcg gtctccacgc atcgtcaggc
     3841 attggcggcc ttgctgttct tctacggcaa ggtgctgtgc acggatctgc cctggcttca
     3901 ggagatcgga agacctcggc cgtcgcggcg cttgccggtg gtgctgaccc cggatgaagt
     3961 ggttcgcatc ctcggttttc tggaaggcga gcatcgtttg ttcgcccagg actctagcta
     4021 tagttctagt ggttggctac taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     4081 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     4141 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     4201 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     4261 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagttt
     4321 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4381 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4441 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4501 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4561 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4621 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4681 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4741 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4801 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4861 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4921 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4981 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5041 tctgtggata accgtattac cgctagcatg gatctcgggg acgtctaact actaagcgag
     5101 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcggaagac tgggcctttc
     5161 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     5221 atttgaacgt tgtgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     5281 ccaggcatca aactaagcag aaggccatc
//
