LOCUS Exported 4706 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Tet inducible microRNA and cDNA expression. ACCESSION . VERSION . KEYWORDS pTRE-T2-miR-HYGRO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4706) TITLE Wright lab backbones REFERENCE 2 (bases 1 to 4706) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4706 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 79..96 /label=L4440 /note="L4440 vector, forward primer" protein_bind 213..234 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 249..279 /label=lac promoter /note="promoter for the E. coli lac operon" promoter 288..602 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 296..314 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 332..350 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 367..385 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 403..421 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 439..457 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 474..492 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 510..528 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 575..599 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind 623..642 /label=T7 /note="T7 promoter, forward primer" promoter 623..641 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 671..695 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" ncRNA 728..754 /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA 796..837 /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" protein_bind complement(866..890) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" polyA_signal 1022..1103 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1059..1078) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1206..1228 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1266..1284) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1352..1456 /gene="bla" /label=AmpR promoter CDS 1457..2317 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGARGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1675..1694) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter 2513..2870 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2536..2556) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2721..2856 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2783..2802 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2901..3938 /codon_start=1 /product="hygromycin B phosphotransferase" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDVTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK EVGRV" RBS 3917..3925 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" polyA_signal 3962..4010 /note="synthetic polyadenylation signal" rep_origin 4042..4631 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4532..4551 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 61 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 121 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 181 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 241 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggctggag tttactccct 301 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgatgtcga 361 gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag 421 aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt ttatccctat 481 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag 541 gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg 601 cctggagaat tcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagt 661 taagctatca acaagtttgt acaaaaaagc aggctttaaa gggaggtagt gagtcgacca 721 gtggatcctg gaggcttgct gaaggctgta tgctggagac cgatcaatcg atcaatcgat 781 caatcggtct ctcaggacac aaggcctgtt actagcactc acatggaaca aatggcccag 841 atctggccgc actcgagata tccagaccca gctttcttgt acaaagtggt tgatctagag 901 ggcccgcggc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt 961 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt 1021 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 1081 aaataaagca tttttttcac tgccactaga gttaagccag ccccgacacc cgccaacacc 1141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 1201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 1261 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 1321 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1381 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1441 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1501 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1561 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1621 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1681 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1741 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1801 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1861 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1921 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1981 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 2041 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 2101 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2161 cggtgagcgt ggggctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2221 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2281 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2341 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2401 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccagc tagctctgcg 2461 cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttagctacc ttctgaggcg 2521 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 2581 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 2641 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 2701 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 2761 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 2821 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcgatt 2881 cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc gttgaaaaat 2941 ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag ggcgaagaga 3001 gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg aatagctgcg 3061 ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct gcactaccca 3121 tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc atcagtagac 3181 gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct gtgttacagc 3241 ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc agcggcttcg 3301 gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc atttgcgcca 3361 ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgtcaccgtg tccgccagcg 3421 tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag gtgcgccacc 3481 tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc atcaccgccg 3541 taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc aacatcttct 3601 tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag cgccggcatc 3661 ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc ctggatcagc 3721 tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa ggccgctgcg 3781 atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct cgccggagcg 3841 cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac cgccggccca 3901 gtacacgacc gcgcgctaag gaggtaggtc gagtttaaac cctagaaccg gtcatggccg 3961 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg ttccggccgt 4021 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 4081 aacaaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 4141 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 4201 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 4261 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 4321 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 4381 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 4441 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 4501 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 4561 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 4621 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 4681 ttgctcacat gttctttcct gcgtta //