LOCUS       sequence-365632-        6507 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 YPet marker and PuroR 
            resistance gene and a PPGK-driven rtTA transactivator 
            (5'ITR-A1-pA::PPGK-rtTA-pA::PRPBSA-YPet-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6507)
  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 6507)
  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: pTS2361_Tier3(SB)-rtTA-YPet_PuroR
FEATURES             Location/Qualifiers
     source          1..6507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(26..817,818..886))
                     /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     649..668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(887..991)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1454..1471)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1460..1684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1732..2231
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1767..1790)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2164..2183
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2284..3024
                     /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(3146..3162)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3146..3162)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4155..4174)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4593..4649
                     /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             4668..5261
                     /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     5161..5181
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    5401..5422
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5824..6412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5904..5923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
       61 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
      121 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcggctc ccacgctcac
      181 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
      241 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
      301 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
      361 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
      421 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
      481 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
      541 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
      601 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
      661 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
      721 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
      781 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
      841 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
      901 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
      961 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     1021 tctaagaaac caagatcagc ggccgctaca gttgaagtcg gaagtttaca tacagttaag
     1081 ttggagtcat taaaactcgt ttttcaacta ctccacaaat ttcttgttaa caaacaatag
     1141 ttttggcaag tcagttagga catctacttt gtgcatgaca caagtcattt ttccaacaat
     1201 tgtttacaga cagattattt cacttataat tcactgtatc acaattccag tgggtcagaa
     1261 gtttacatac actaagttcg actcctctgc agaatgcggc gatgtttcgg taaggggtcc
     1321 gctatctagt cttaagagat ctacgcgtgg taccctcgag gagctcttcg aagcggaatt
     1381 caccatgact agtgctagcg gatccaccgg tgtctagaaa gctttgaggc cggcctgcag
     1441 gcccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1501 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1561 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1621 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1681 atggtagctt aggaggcgga aagaaccagc gcgtggtacc ctcgagctac cgggtagggg
     1741 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     1801 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     1861 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     1921 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcagta
     1981 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     2041 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     2101 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     2161 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     2221 cgggcctttc gacctccagc ccgagctcgg tatagggaga ccgaattcac catgactagt
     2281 ggatcaagac tggacaagag caaagtcata aactctgctc tggaattact caatggagtc
     2341 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc
     2401 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg
     2461 gacaggcatc atacccactc ctgccccctg gaaggcgagt catggcaaga ctttctgcgg
     2521 aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat
     2581 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg
     2641 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt
     2701 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca
     2761 cctaccaccg attctatgcc cccacttctg aaacaagcaa ttgagctgtt cgaccggcag
     2821 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag
     2881 ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc
     2941 ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat
     3001 tttgaccttg acatgctccc cggggctagc ggatccaccg gtgtctagaa agctatacag
     3061 gtgcaccgct ttataataaa actgatacag agaacgttat catgtgtgtt ggtttttttt
     3121 gtgtgtgcgg tttaagggag cctcggtcat agctgtttcc tgtgtaaaac gacggccagc
     3181 gcgtggtacc ctcgagcgtg accctgtctc acaaaataaa gtaagcccgg actgagtgcg
     3241 gaaaggcggg cctggcgggt ctggactccc catgcgggcc accagaggcc ctgcagcctt
     3301 cagtcgcttg aaggggtaat ggcgcttcca ctcacaaaca tggcggacag agcgtgtgaa
     3361 cgagatgaac agcccctcaa aaatatggcc gccgaggctg gacggccgtg ccccagcagc
     3421 accgcctccg cgccccacgt gatctctcgc cgggcacagc gctgaccgcg gaggtccaac
     3481 cggaagaatg tccggattgg acattcggaa gagggcccgc cttccctggg gaatctctgc
     3541 gcacgcgcag aacgcttcga ccaatgaaaa cacaggaagc cgtccgcgca accgcgttgc
     3601 gtcacttctg ccgcccctgt ttcaaggtat atagccgtag acggaacttc gcctttctct
     3661 cggccttagc gccatttttt tgggtgagtg ttttttggtt cctgcgttgg gattccgtgt
     3721 acaatccata gacatctgac ctcggcactt agcatcatca cagcaaacta actgtagcct
     3781 ttctctcttt ccctgtagaa acctctgcac ctagggagct cgaattcacc atgactagtg
     3841 gtggttctgg tgtgagcaaa ggcgaagagc tgttcaccgg cgtggtgccc atcctggtgg
     3901 agctggacgg cgacgtgaac ggccacaagt tcagcgtgag cggcgagggc gagggcgacg
     3961 ccacctacgg caagctgacc ctgaagctgc tgtgcaccac cggcaagctg cccgtgccct
     4021 ggcccaccct ggtgaccacc ctgggctacg gcgtgcagtg cttcgcccgg taccccgacc
     4081 acatgaagca gcacgacttc ttcaagagcg ccatgcccga gggctacgtg caggagcgga
     4141 ccatcttctt caaggacgac ggcaactaca agacccgggc cgaggtgaag ttcgagggcg
     4201 acaccctggt gaaccggatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     4261 tgggccacaa gctggagtac aactacaaca gccacaacgt gtacatcacc gccgacaagc
     4321 agaagaacgg catcaaggcc aacttcaaga tccggcacaa catcgaggac ggcggcgtgc
     4381 agctggccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     4441 acaaccacta cctgagctac cagagcgccc tgttcaagga ccccaacgag aagcgggacc
     4501 acatggtgct gctggagttc ctgaccgccg ccggcatcac cgagggcatg gacgagctgt
     4561 acaaggctag tggtggttct ggtggaagcg gagctactaa cttcagcctg ctgaagcagg
     4621 ctggtgacgt ggaggagaac cctggacctg ctagtggtgg ttctggtacc gagtacaagc
     4681 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     4741 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg
     4801 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     4861 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     4921 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     4981 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     5041 ccaccgtcgg cgtgtcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     5101 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca tccgcgcccc
     5161 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     5221 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cgctagcgga tccaccggtg
     5281 tctagaaagc tcagaaatta tgagagcgtt taataaagta ttgactatgg ttgacaaaac
     5341 gtgtgttgga gttttttgtg tgctttgtca tcaactgagc cagtaggtcg agggtgccta
     5401 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5461 cctgtcgtgc cagctgctag ctactcggga ccccttaccg aaacatcgcc gcattctgca
     5521 gaggagtcga gtgtatgtaa acttctgacc cactgggaat gtgatgaaag aaataaaagc
     5581 tgaaatgaat cattctctct actattattc tgatatttca cattcttaaa ataaagtggt
     5641 gatcctaact gacctaagac agggaatttt tactaggatt aaatgtcagg aattgtgaaa
     5701 aagtgagttt aaatgtattt ggctaaggtg tatgtaaact tccgacttca actgtaccat
     5761 ggacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5821 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5881 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5941 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6001 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6061 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6121 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6181 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6241 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6301 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6361 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6421 tttgatcttt tctacgggga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6481 aatgaagttt taaatcaatc taaagta
//
