LOCUS Exported 6047 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Tol2 doxycycline inducible expression vector. ACCESSION . VERSION . KEYWORDS pAK_Tol2_TRE_Zeo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6047) TITLE Unpublished JOURNAL Unpublished REFERENCE 2 (bases 1 to 6047) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6047 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 284..598 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 292..310 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 328..346 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 363..381 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 399..417 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 435..453 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 470..488 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 506..524 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 571..595 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind complement(683..700) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 689..913 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 963..1084 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(974..993) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1028..1047 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(1193..1939) /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS complement(1940..1993) /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS complement(1994..2365) /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" promoter complement(2375..2885) /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind complement(2438..2456) /label=hPGK-F /note="Human PGK promoter, forward primer" promoter complement(3159..3177) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3160..3177) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3195..3211) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3195..3211) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3208..3230) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3219..3235 /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)." promoter complement(3243..3273) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3288..3309 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3426..3443) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3597..4185) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3677..3696) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4356..5216) /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 4979..4998 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5217..5321) /gene="bla" /label=AmpR promoter rep_origin complement(5399..5854) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5536..5557) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5748..5767 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5980..6002 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5994..6011 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5995..6011 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 6018..6037 /label=T7 /note="T7 promoter, forward primer" promoter 6018..6036 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 ccagaggtgt aaagtacttg agtaatttta cttgattact gtacttaagt attatttttg 61 gggattttta ctttacttga gtacaattaa aaatcaatac ttttactttt acttaattac 121 atttttttag aaaaaaaagt actttttact ccttacaatt ttatttacag tcaaaaagta 181 cttatttttt ggagatcact tcattctatt ttcccttgct attaccaaac caattgaatt 241 gcgctgatgc ccagtttaat ttaaatagat ctatcgatat gtcgagttta ctccctatca 301 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacga tgtcgagttt 361 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg 421 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttat ccctatcagt 481 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgaggtag 541 gcgtgtacgg tgggaggcct atataagcag agctcgttta gtgaaccgtc agatcgcctg 601 gagaattggc tagctagaca ggatccacgc gtggccgcgc gcactagtga attccatgga 661 tatctttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 721 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 781 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 841 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 901 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtaatcaagc 961 tttaagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 1021 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 1081 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt 1141 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc agttacccgg 1201 ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt 1261 cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc 1321 cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa 1381 ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtctc agaagtgggg 1441 gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgttcct 1501 ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca 1561 gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct 1621 ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg agagcacagc 1681 ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcaga 1741 agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg gcccgcttgt 1801 tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg 1861 tcgtcaggcc ttcgataccg acaccattga gtaattccag agctccgttt atgactttgc 1921 tcttgtccag tctagacatt ggaccagggt tttcttcaac atcaccacaa gtgaggagag 1981 aacctctacc ttcgtcctgc tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc 2041 gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc ggcccggagg 2101 cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg tccaggccgc 2161 gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga 2221 acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag tcccgggaga 2281 acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg 2341 gaacggcact ggtcaacttg gccatggtga attgctgggg agagaggtcg gtgattcggt 2401 caacgaggga gccgactgcc gacgtgcgct ccggaggctt gcagaatgcg gaacaccgcg 2461 cgggcaggaa cagggcccac actaccgccc cacaccccgc ctcccgcacc gccccttccc 2521 ggccgctgct ctcggcgcgc cctgctgagc agccgctatt ggccacagcc catcgcggtc 2581 ggcgcgctgc cattgctccc tggcgctgtc cgtctgcgag ggtactagtg agacgtgcgg 2641 cttccgtttg tcacgtccgg cacgccgcga accgcaagga accttcccga cttaggggcg 2701 gagcaggaag cgtcgccggg gggcccacaa gggtagcggc gaagatccgg gtgacgctgc 2761 gaacggacgt gaagaatgtg cgagacccag ggtcggcgcc gctgcgtttc ccggaaccac 2821 gcccagagca gccgcgtccc tgcgcaaacc cagggctgcc ttggaaaagg cgcaacccca 2881 acccctaaac aagaatctct agttttcttt cttgctttta cttttacttc cttaatactc 2941 aagtacaatt ttaatggagt acttttttac ttttactcaa gtaagattct agccagatac 3001 ttttactttt aattgagtaa aattttccct aagtacttgt actttcactt gagtaaaatt 3061 tttgagtact ttttacacct ctgctcgacc atatgggaga gctcccaacg cgatagatat 3121 gataattgga atgcgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc 3181 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 3241 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 3301 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3361 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3421 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3481 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3541 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3601 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3661 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3721 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3781 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3841 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3901 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3961 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 4021 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 4081 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 4141 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4201 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 4261 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 4321 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 4381 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 4441 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 4501 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 4561 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 4621 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 4681 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 4741 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 4801 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 4861 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 4921 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 4981 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 5041 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 5101 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 5161 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 5221 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 5281 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 5341 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa 5401 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt 5461 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata 5521 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac 5581 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa 5641 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc 5701 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg 5761 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca 5821 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact 5881 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat 5941 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 6001 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggc //