LOCUS Exported 4422 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Entry vector for cloning miR30-based shRNA driven by TRE-tight promoter.. ACCESSION . VERSION . KEYWORDS pEN_TTmiRc2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4422) 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 4422) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4422 /organism="synthetic DNA construct" /mol_type="other DNA" 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" promoter 198..512 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 206..224 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 242..260 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 277..295 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 313..331 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 349..367 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 384..402 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 420..438 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 485..509 /label=LNCX /note="Human CMV promoter, forward primer" ncRNA 544..665 /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 701..1006 /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 885..904 /label=ccdB-fwd /note="ccdB gene, forward primer" ncRNA 1027..1153 /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(1192..1291) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1403..1422) /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind complement(1481..1500) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" CDS complement(2074..2607) /codon_start=1 /gene="aacC1" /product="gentamycin acetyltransferase" /label=GmR /note="confers resistance to gentamycin" /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR EEVMHFDIDPSTAT" promoter complement(2796..2824) /gene="intI1 (promoter lies within the coding sequence)" /label=Pc promoter /note="class 1 integron promoter" rep_origin 3481..4069 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3970..3989 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 4234..4320 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4412..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 ggatcccgag tttactccct atcagtgata gagaacgtat gtcgagttta 241 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta 301 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg 361 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact 421 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa 481 gcagagctcg tttagtgaac cgtcagatcg ccactagtcg actagggata acagggtaat 541 tgtttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg gaaacacttg 601 ctgggattac ttcttcaggt taacccaaca gaaggctcga gaaggtatat tgctgttgac 661 agtgagcgtg ctgcaggtaa gcttgaggtg tgcttccaga atgcagttta aggtttacac 721 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac 781 gcccgggcga cggatagtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc 841 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga 901 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga 961 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atatagaatt ccaacctgct 1021 gcttgcctac tgcctcggaa ttcaaggggc tactttagga gcaattatct tgtttactaa 1081 aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa aatggtataa 1141 attaaatcac ttttttcaat tggaagacta atgcgttttc gagatatcta gacccagctt 1201 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg 1261 tcactatcag tcaaaataaa atcattattt gccatccagc tgcagctctg gcccgtgtct 1321 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg 1381 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg 1441 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat 1501 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag 1561 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga 1621 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct 1681 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa 1741 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgtccct gcgccggttg 1801 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag 1861 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat 1921 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat 1981 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta 2041 ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg gcggtacttg ggtcgatatc 2101 aaagtgcatc acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc 2161 accgtaatct gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag 2221 gagattgatg agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc 2281 atagatatag atctcactac gcggctgctc aaacctgggc agaacgtaag ccgcgagagc 2341 gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg aatgtcttac tacggagcaa 2401 gttcccgagg taatcggagt ccggctgatg ttgggagtag gtggctacgt ctccgaactc 2461 acgaccgaaa agatcaagag cagcccgcat ggatttgact tggtcagggc cgagcctaca 2521 tgtgcgaatg atgcccatac ttgagccacc taactttgtt ttagggcgac tgccctgctg 2581 cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca 2641 cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa aaacagtcat 2701 aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg 2761 accagttgcg tgagcgcata cgctacttgc attacagttt acgaaccgaa caggcttatg 2821 tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg tcacccggca accttgggca 2881 gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca 2941 cgcatcgtca ggcattggcg gccttgctgt tcttctacgg caaggtgctg tgcacggatc 3001 tgccctggct tcaggagatc ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga 3061 ccccggatga agtggttcgc atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc 3121 aggactctag ctatagttct agtggttggc tactaatagg ttgtattgat gttggacgag 3181 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 3241 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 3301 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt 3361 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc 3421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 3481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 3541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 3601 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 3661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 3721 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 3781 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 3841 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 3901 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 3961 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 4021 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 4081 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 4141 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta 4201 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa 4261 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat 4321 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc 4381 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tc //