LOCUS       Exported                7433 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 neomycin
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    pSBtet-Neo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7433)
  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 7433)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7433
                     /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..4819
                     /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(4079..4098)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4689..4708
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4874..4891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4880..5104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5430..5447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5601..6189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5681..5700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6360..7220)
                     /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     6983..7002
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7221..7325)
                     /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 tcccatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4081 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4141 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4201 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4261 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4321 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4381 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4441 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4501 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4561 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4621 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4681 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4741 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4801 tcttgacgag ttcttctgat tcgaaggcct gtcgtgaagc ttggggatca attctctaga
     4861 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4921 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4981 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5041 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     5101 atggcttctg aggcggaaag aaccagctgg gcctactagc tactcgggac cccttaccga
     5161 aacatcgccg cattctgcag aggagtcgag tgtatgtaaa cttctgaccc actgggaatg
     5221 tgatgaaaga aataaaagct gaaatgaatc attctctcta ctattattct gatatttcac
     5281 attcttaaaa taaagtggtg atcctaactg acctaagaca gggaattttt actaggatta
     5341 aatgtcagga attgtgaaaa agtgagttta aatgtatttg gctaaggtgt atgtaaactt
     5401 ccgacttcaa ctgtataggg atccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5461 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5521 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5581 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5641 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5701 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5761 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5821 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5881 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5941 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6001 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6061 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6121 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6181 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6241 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6301 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6361 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6421 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6481 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6541 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6601 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6661 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6721 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6781 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6841 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6901 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6961 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7021 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7081 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7141 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7201 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7261 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7321 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     7381 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa att
//
