LOCUS       sequence-365631-        5691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable stable integration of up to three cassettes
            via sleeping beauty transposase including a PuroR resistance gene 
            and a PPGK-driven rtTA transactivator 
            (5'ITR-A1-pA::PPGK-rtTA-pA::PRPBSA-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5691)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 5691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pTS2362_Tier3(SB)-rtTA-PuroR
FEATURES             Location/Qualifiers
     source          1..5691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(50..841,842..910))
                     /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     673..692
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(911..1015)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1478..1495)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1484..1708
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1756..2255
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1791..1814)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2188..2207
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2308..3048
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="SRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTD
                     SMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(3170..3186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3170..3186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3876..4469
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4369..4389
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4609..4630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5032..5620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5112..5131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
       61 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      121 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      181 tgataccgcg gctcccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
      241 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
      301 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      361 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      421 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
      481 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
      541 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
      601 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      661 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      721 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      781 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      841 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      901 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
      961 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1021 ttccccgaaa agtgccacct gacgtctaag aaaccaagat cagcggccgc tacagttgaa
     1081 gtcggaagtt tacatacagt taagttggag tcattaaaac tcgtttttca actactccac
     1141 aaatttcttg ttaacaaaca atagttttgg caagtcagtt aggacatcta ctttgtgcat
     1201 gacacaagtc atttttccaa caattgttta cagacagatt atttcactta taattcactg
     1261 tatcacaatt ccagtgggtc agaagtttac atacactaag ttcgactcct ctgcagaatg
     1321 cggcgatgtt tcggtaaggg gtccgctatc tagtcttaag agatctacgc gtggtaccct
     1381 cgaggagctc ttcgaagcgg aattcaccat gactagtgct agcggatcca ccggtgtcta
     1441 gaaagctttg aggccggcct gcaggcccgc tgatcagcct cgactgtgcc ttctagttgc
     1501 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1561 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1621 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1681 catgctgggg atgcggtggg ctctatggta gcttaggagg cggaaagaac cagcgcgtgg
     1741 taccctcgag ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     1801 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     1861 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     1921 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     1981 aaatggaagt agcacgtctc agtagtctcg tgcagatgga cagcaccgct gagcaatgga
     2041 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     2101 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     2161 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     2221 cgctgttctc ctcttcctca tctccgggcc tttcgacctc cagcccgagc tcggtatagg
     2281 gagaccgaat tcaccatgac tagtggatca agactggaca agagcaaagt cataaactct
     2341 gctctggaat tactcaatgg agtcggtatc gaaggcctga cgacaaggaa actcgctcaa
     2401 aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc
     2461 gatgccctgc caatcgagat gctggacagg catcataccc actcctgccc cctggaaggc
     2521 gagtcatggc aagactttct gcggaacaac gccaagtcat accgctgtgc tctcctctca
     2581 catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc
     2641 ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac
     2701 gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa
     2761 gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact tctgaaacaa
     2821 gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta
     2881 atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgac cgacgccctt
     2941 gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg
     3001 ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccggggc tagcggatcc
     3061 accggtgtct agaaagctat acaggtgcac cgctttataa taaaactgat acagagaacg
     3121 ttatcatgtg tgttggtttt ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt
     3181 ttcctgtgta aaacgacggc cagcgcgtgg taccctcgag cgtgaccctg tctcacaaaa
     3241 taaagtaagc ccggactgag tgcggaaagg cgggcctggc gggtctggac tccccatgcg
     3301 ggccaccaga ggccctgcag ccttcagtcg cttgaagggg taatggcgct tccactcaca
     3361 aacatggcgg acagagcgtg tgaacgagat gaacagcccc tcaaaaatat ggccgccgag
     3421 gctggacggc cgtgccccag cagcaccgcc tccgcgcccc acgtgatctc tcgccgggca
     3481 cagcgctgac cgcggaggtc caaccggaag aatgtccgga ttggacattc ggaagagggc
     3541 ccgccttccc tggggaatct ctgcgcacgc gcagaacgct tcgaccaatg aaaacacagg
     3601 aagccgtccg cgcaaccgcg ttgcgtcact tctgccgccc ctgtttcaag gtatatagcc
     3661 gtagacggaa cttcgccttt ctctcggcct tagcgccatt tttttgggtg agtgtttttt
     3721 ggttcctgcg ttgggattcc gtgtacaatc catagacatc tgacctcggc acttagcatc
     3781 atcacagcaa actaactgta gcctttctct ctttccctgt agaaacctct gcacctaggg
     3841 agctcgaatt caccatgact agtggtggtt ctggtaccga gtacaagccc acggtgcgcc
     3901 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     3961 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     4021 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     4081 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     4141 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     4201 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     4261 tgtcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     4321 cggccgagcg cgccggggtg cccgccttcc tggagacatc cgcgccccgc aacctcccct
     4381 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     4441 cctggtgcat gacccgcaag cccggtgccg ctagcggatc caccggtgtc tagaaagctc
     4501 agaaattatg agagcgttta ataaagtatt gactatggtt gacaaaacgt gtgttggagt
     4561 tttttgtgtg ctttgtcatc aactgagcca gtaggtcgag ggtgcctaat gagtgagcta
     4621 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4681 gctgctagct actcgggacc ccttaccgaa acatcgccgc attctgcaga ggagtcgagt
     4741 gtatgtaaac ttctgaccca ctgggaatgt gatgaaagaa ataaaagctg aaatgaatca
     4801 ttctctctac tattattctg atatttcaca ttcttaaaat aaagtggtga tcctaactga
     4861 cctaagacag ggaattttta ctaggattaa atgtcaggaa ttgtgaaaaa gtgagtttaa
     4921 atgtatttgg ctaaggtgta tgtaaacttc cgacttcaac tgtaccatgg acatgtgagc
     4981 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5041 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5101 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5161 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5221 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5281 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5401 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5461 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5521 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5581 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5641 tacggggatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa t
//
