LOCUS Exported 7037 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION SB-transposon with inducible SfiI cloning site for GOI (contains firefly luciferase) and constitutive expression of rtTA and blasticidin resistance gene. ACCESSION . VERSION . KEYWORDS pSBtet-Bla SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7037) AUTHORS Kowarz E, Loescher D, Marschalek R TITLE Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. JOURNAL Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821. PUBMED 25650551 REFERENCE 2 (bases 1 to 7037) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7037 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 295..607 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 303..321 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 339..357 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 374..392 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 410..428 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 446..464 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 481..499 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 517..535 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 580..604 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 680..689 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 686..2338 /codon_start=1 /product="firefly luciferase" /label=luciferase /note="synthetic luc2 version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" polyA_signal 2374..2495 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2385..2404) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2439..2458 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS 3218..3961 /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 KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS 3968..4024 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 4025..4423 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" primer_bind complement(4478..4495) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4484..4708 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5034..5051) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5205..5793) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5285..5304) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5964..6824) /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 6587..6606 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6825..6929) /gene="bla" /label=AmpR promoter ORIGIN 1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact 61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta 121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta 181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt 241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt 301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg 361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt 421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta 481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta 541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca 601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta 661 ccctcgaaag gcctctgagg ccaccatgga agatgccaaa aacattaaga agggcccagc 721 gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg 781 ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg tggacattac 841 ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc gctatgggct 901 gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt 961 gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct acaacgagcg 1021 cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg 1081 gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat 1141 ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga cttcccattt 1201 gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg acaaaaccat 1261 cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag ccctaccgca 1321 ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca accagatcat 1381 ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac 1441 gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct 1501 attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt 1561 tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat 1621 cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca 1681 cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac 1741 ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa 1801 ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt 1861 ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat 1921 cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt 1981 cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc 2041 cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct 2101 gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac 2161 catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct 2221 gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc 2281 ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg ccgtgtaagg 2341 cctgtcaggc caagcttcca tcgatagaca tgataagata cattgatgag tttggacaaa 2401 ccacaacaag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt 2461 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta 2521 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat 2581 gtggtaccta ggcgagaccc tgtctcacaa aataaagtaa gcccggactg agtgcggaaa 2641 ggcgggcctg gcgggtctgg tctccccatg cgggccacca gaggccctgc agccttcagt 2701 cgcttgaagg ggtaatggcg cttccactca caaacatggc ggacagagcg tgtgaacgag 2761 atgaacagcc cctcaaaaat atggccgccg aggctggacg gccgtgcccc agcagcaccg 2821 cctccgcgcc ccacgtgatc tctcgccggg cacagcgctg accgcggagg tccaaccgga 2881 agaatgtccg gattggacat tcggaagagg gcccgccttc cctggggaat ctctgcgcac 2941 gcgcagaacg cttcgaccaa tgaaaacaca ggaagccgtc cgcgcaaccg cgttgcgtca 3001 cttctgccgc ccctgtttca aggtatatag ccgtagacgg aacttcgcct ttctctcggc 3061 cttagcgcca tttttttggg tgagtgtttt ttggttcctg cgttgggatt ccgtgtacaa 3121 tccatagaca tctgacctcg gcacttagca tcatcacagc aaactaactg tagcctttct 3181 ctctttccct gtagaaacct ctgcacctga ggccaccatg tctagactgg acaagagcaa 3241 agtcataaac ggcgctctgg aattactcaa tggagtcggt atcgaaggcc tgacgacaag 3301 gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa 3361 gcgggccctg ctcgatgccc tgccaatcga gatgctggac aggcatcata cccacttctg 3421 ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cattccgctg 3481 tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa 3541 acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga 3601 gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga 3661 acaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc 3721 acttctgaga caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt 3781 cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc 3841 ggccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt 3901 tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg 3961 gtccggcgct actaacttca gcctgctgaa gcaggctggt gacgtcgagg agaatcctgg 4021 tcccatggcc atgcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac 4081 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga 4141 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact 4201 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat 4261 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga 4321 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg 4381 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taattcgaag gcctgtcgtg 4441 aagcttgggg atcaattctc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc 4501 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 4561 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 4621 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 4681 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctgggcctac 4741 tagctactcg ggacccctta ccgaaacatc gccgcattct gcagaggagt cgagtgtatg 4801 taaacttctg acccactggg aatgtgatga aagaaataaa agctgaaatg aatcattctc 4861 tctactatta ttctgatatt tcacattctt aaaataaagt ggtgatccta actgacctaa 4921 gacagggaat ttttactagg attaaatgtc aggaattgtg aaaaagtgag tttaaatgta 4981 tttggctaag gtgtatgtaa acttccgact tcaactgtat agggatccgc ttcctcgctc 5041 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 5101 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 5161 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 5221 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 5281 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 5341 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 5401 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 5461 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 5521 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 5581 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 5641 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 5701 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 5761 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5821 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5881 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5941 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 6001 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 6061 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 6121 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 6181 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 6241 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 6301 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 6361 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 6421 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 6481 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 6541 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 6601 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 6661 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6721 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6781 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6841 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6901 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg 6961 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 7021 aatattttgt taaaatt //