LOCUS       Exported                7802 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 BFP, rtTA and 
            blasticidin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    pSBtet-BB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7802)
  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 7802)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7802
                     /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             3926..3982
                     /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             3983..4726
                     /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             4733..4789
                     /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             4790..5188
                     /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(5243..5260)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5249..5473
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5799..5816)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5970..6558)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6050..6069)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6729..7589)
                     /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     7352..7371
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7590..7694)
                     /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 agcgagctga ttaaggagaa
     3241 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     3301 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     3361 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     3421 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     3481 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     3541 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     3601 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     3661 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     3721 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     3781 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     3841 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     3901 ggggcacaag ctgaacggca gtggagctac taacttcagc ctgctgaagc aggctggtga
     3961 cgtcgaggag aatcctggcc ccatgtctag actggacaag agcaaagtca taaacggcgc
     4021 tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa
     4081 gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga
     4141 tgccctgcca atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga
     4201 gtcatggcaa gactttctgc ggaacaacgc caagtcattc cgctgtgctc tcctctcaca
     4261 tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct
     4321 ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc
     4381 tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt
     4441 agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc
     4501 aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat
     4561 catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccggccg acgcccttga
     4621 cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct
     4681 gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtccg gcgctactaa
     4741 cttcagcctg ctgaagcagg ctggtgacgt cgaggagaat cctggtccca tggccatgcc
     4801 tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc
     4861 catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac
     4921 tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac
     4981 tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     5041 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     5101 caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct
     5161 gccctctggt tatgtgtggg agggctaatt cgaaggcctg tcgtgaagct tggggatcaa
     5221 ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     5281 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     5341 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     5401 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     5461 gtgggctcta tggcttctga ggcggaaaga accagctggg cctactagct actcgggacc
     5521 ccttaccgaa acatcgccgc attctgcaga ggagtcgagt gtatgtaaac ttctgaccca
     5581 ctgggaatgt gatgaaagaa ataaaagctg aaatgaatca ttctctctac tattattctg
     5641 atatttcaca ttcttaaaat aaagtggtga tcctaactga cctaagacag ggaattttta
     5701 ctaggattaa atgtcaggaa ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta
     5761 tgtaaacttc cgacttcaac tgtataggga tccgcttcct cgctcactga ctcgctgcgc
     5821 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5881 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5941 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6001 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6061 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6121 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6181 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6241 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6301 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6361 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6421 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6481 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6541 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6601 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6661 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6721 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6781 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6841 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6901 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6961 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7021 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7081 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7141 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7201 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7261 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7321 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7381 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7441 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7501 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7561 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7621 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7681 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca
     7741 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     7801 tt
//
