pSBtet-Hyg

LOCUS      pSBtet-Hyg               7664 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 
            hygromycin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    pSBtet-Hyg
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7664)
  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 7664)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7664
                     /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..5050
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5105..5122)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5111..5335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5661..5678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5832..6420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5912..5931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6591..7451)
                     /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     7214..7233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7452..7556)
                     /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 tcccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     4081 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     4141 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     4201 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     4261 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     4321 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     4381 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     4441 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     4501 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     4561 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     4621 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     4681 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     4741 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     4801 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     4861 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     4921 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     4981 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     5041 aaaggaatag ttcgaaggcc tgtcgtgaag cttggggatc aattctctag agctcgctga
     5101 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5161 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5221 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5281 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     5341 gaggcggaaa gaaccagctg ggcctactag ctactcggga ccccttaccg aaacatcgcc
     5401 gcattctgca gaggagtcga gtgtatgtaa acttctgacc cactgggaat gtgatgaaag
     5461 aaataaaagc tgaaatgaat cattctctct actattattc tgatatttca cattcttaaa
     5521 ataaagtggt gatcctaact gacctaagac agggaatttt tactaggatt aaatgtcagg
     5581 aattgtgaaa aagtgagttt aaatgtattt ggctaaggtg tatgtaaact tccgacttca
     5641 actgtatagg gatccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5701 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5761 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5821 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5881 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5941 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6001 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6061 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6121 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6181 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6241 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6301 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6361 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6421 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6481 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6541 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6601 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6661 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6721 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6781 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6841 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6901 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6961 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7021 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7081 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7141 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7201 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7261 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7321 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7381 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7441 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7501 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7561 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
     7621 ataccgcatc aggaaattgt aagcgttaat attttgttaa aatt
//