LOCUS Exported 8201 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 RFP, rtTA and neomycin resistance gene. ACCESSION . VERSION . KEYWORDS pSBtet-RN SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8201) 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 8201) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8201 /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..3919 /codon_start=1 /product="dimeric variant of DsRed fluorescent protein (Shaner et al., 2004)" /label=dTomato /note="mammalian codon-optimized" /translation="MLSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH HLFLYGMDELYK" primer_bind complement(3351..3369) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3619..3638 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3813..3836 /label=DsRed1-C /note="DsRed1, forward primer" CDS 3929..3985 /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 3986..4729 /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 4736..4792 /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 4793..5587 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4847..4866) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5457..5476 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5642..5659) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5648..5872 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6198..6215) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6369..6957) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6449..6468) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7128..7988) /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 7751..7770 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7989..8093) /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 ttgagcaagg gcgaggaggt 3241 catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg gccatgagtt 3301 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa 3361 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcatgta 3421 cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga agctgtcctt 3481 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt 3541 gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga tgcgcggcac 3601 caacttcccc cccgacggcc ccgtaatgca gaagaagaca atgggctggg aggcctccac 3661 cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg ccctgaagct 3721 gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca agaagcccgt 3781 gcaactgccc ggctactact acgtggacac caagctggac atcacctccc acaacgagga 3841 ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt tcctgtacgg 3901 catggacgag ctgtacaagg gcagtggagc tactaacttc agcctgctga agcaggctgg 3961 tgacgtcgag gagaatcctg gccccatgtc tagactggac aagagcaaag tcataaacgg 4021 cgctctggaa ttactcaatg gagtcggtat cgaaggcctg acgacaagga aactcgctca 4081 aaagctggga gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct 4141 cgatgccctg ccaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg 4201 cgagtcatgg caagactttc tgcggaacaa cgccaagtca ttccgctgtg ctctcctctc 4261 acatcgcgac ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac 4321 cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactgta 4381 cgctctgtcc gccgtgggcc actttacact gggctgcgta ttggaggaac aggagcatca 4441 agtagcaaaa gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca 4501 agcaattgag ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg gcctggaact 4561 aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc ggcgggccgg ccgacgccct 4621 tgacgatttt gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat 4681 gctgcctgct gacgctcttg acgattttga ccttgacatg ctccccgggt ccggcgctac 4741 taacttcagc ctgctgaagc aggctggtga cgtcgaggag aatcctggtc ccatgattga 4801 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 4861 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 4921 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga 4981 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 5041 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 5101 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 5161 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 5221 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 5281 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga 5341 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 5401 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 5461 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 5521 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 5581 cttctgattc gaaggcctgt cgtgaagctt ggggatcaat tctctagagc tcgctgatca 5641 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 5701 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 5761 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 5821 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 5881 gcggaaagaa ccagctgggc ctactagcta ctcgggaccc cttaccgaaa catcgccgca 5941 ttctgcagag gagtcgagtg tatgtaaact tctgacccac tgggaatgtg atgaaagaaa 6001 taaaagctga aatgaatcat tctctctact attattctga tatttcacat tcttaaaata 6061 aagtggtgat cctaactgac ctaagacagg gaatttttac taggattaaa tgtcaggaat 6121 tgtgaaaaag tgagtttaaa tgtatttggc taaggtgtat gtaaacttcc gacttcaact 6181 gtatagggat ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 6241 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 6301 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 6361 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 6421 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 6481 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 6541 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 6601 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 6661 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 6721 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 6781 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 6841 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 6901 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 6961 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 7021 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 7081 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 7141 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 7201 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 7261 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 7321 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 7381 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 7441 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 7501 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 7561 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 7621 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 7681 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 7741 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 7801 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 7861 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 7921 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 7981 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 8041 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 8101 ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 8161 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat t //