LOCUS       sequence-366786-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable integration of up to three cassettes via 
            sleeping beauty transposase including a TagBFP marker and PuroR 
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-mTagBFP2-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  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 5185)
  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: pTS2337_Tier3(SB)-TagBFP2_Puro
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     619..638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(857..961)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1424..1441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1430..1654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1800..1816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1800..1816)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2506..3213
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3241..3297
                     /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             3316..3909
                     /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     3809..3829
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4049..4070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4472..5060)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4552..4571)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5181..5185,1..787,788..856))
                     /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"
ORIGIN
        1 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
       61 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      121 ctgcaatgat accgcggctc ccacgctcac cggctccaga tttatcagca ataaaccagc
      181 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      241 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      301 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
      361 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
      421 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
      481 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
      541 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
      601 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
      661 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
      721 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
      781 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
      841 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
      901 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
      961 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac caagatcagc ggccgctaca
     1021 gttgaagtcg gaagtttaca tacagttaag ttggagtcat taaaactcgt ttttcaacta
     1081 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt
     1141 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat
     1201 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttcg actcctctgc
     1261 agaatgcggc gatgtttcgg taaggggtcc gctatctagt cttaagagat ctacgcgtgg
     1321 taccctcgag gagctcttcg aagcggaatt caccatgact agtgctagcg gatccaccgg
     1381 tgtctagaaa gctttgaggc cggcctgcag gcccgctgat cagcctcgac tgtgccttct
     1441 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1501 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     1561 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     1621 agcaggcatg ctggggatgc ggtgggctct atggtagctt aggaggcgga aagaaccagc
     1681 gcgggagacc gtcagttggt ctccagctat acaggtgcac cgctttataa taaaactgat
     1741 acagagaacg ttatcatgtg tgttggtttt ttttgtgtgt gcggtttaag ggagcctcgg
     1801 tcatagctgt ttcctgtgta aaacgacggc cagcgcgtgg taccctcgag cgtgaccctg
     1861 tctcacaaaa taaagtaagc ccggactgag tgcggaaagg cgggcctggc gggtctggac
     1921 tccccatgcg ggccaccaga ggccctgcag ccttcagtcg cttgaagggg taatggcgct
     1981 tccactcaca aacatggcgg acagagcgtg tgaacgagat gaacagcccc tcaaaaatat
     2041 ggccgccgag gctggacggc cgtgccccag cagcaccgcc tccgcgcccc acgtgatctc
     2101 tcgccgggca cagcgctgac cgcggaggtc caaccggaag aatgtccgga ttggacattc
     2161 ggaagagggc ccgccttccc tggggaatct ctgcgcacgc gcagaacgct tcgaccaatg
     2221 aaaacacagg aagccgtccg cgcaaccgcg ttgcgtcact tctgccgccc ctgtttcaag
     2281 gtatatagcc gtagacggaa cttcgccttt ctctcggcct tagcgccatt tttttgggtg
     2341 agtgtttttt ggttcctgcg ttgggattcc gtgtacaatc catagacatc tgacctcggc
     2401 acttagcatc atcacagcaa actaactgta gcctttctct ctttccctgt agaaacctct
     2461 gcacctaggg agctcgaatt caccatgact agtggtggtt ctggtgtgtc taagggcgaa
     2521 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     2581 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     2641 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     2701 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     2761 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     2821 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     2881 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     2941 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     3001 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa
     3061 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     3121 aaggaggcca acaacgaaac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     3181 gacctcccta gcaaactggg gcacaaactt aatgctagtg gtggttctgg tggaagcgga
     3241 gctactaact tcagcctgct gaagcaggct ggtgacgtgg aggagaaccc tggacctgct
     3301 agtggtggtt ctggtaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     3361 ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     3421 gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     3481 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     3541 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3601 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3661 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tgtcgcccga ccaccagggc
     3721 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3781 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3841 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3901 cccggtgccg ctagcggatc caccggtgtc tagaaagctc agaaattatg agagcgttta
     3961 ataaagtatt gactatggtt gacaaaacgt gtgttggagt tttttgtgtg ctttgtcatc
     4021 aactgagcca gtaggtcgag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4081 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgctagct actcgggacc
     4141 ccttaccgaa acatcgccgc attctgcaga ggagtcgagt gtatgtaaac ttctgaccca
     4201 ctgggaatgt gatgaaagaa ataaaagctg aaatgaatca ttctctctac tattattctg
     4261 atatttcaca ttcttaaaat aaagtggtga tcctaactga cctaagacag ggaattttta
     4321 ctaggattaa atgtcaggaa ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta
     4381 tgtaaacttc cgacttcaac tgtaccatgg acatgtgagc aaaaggccag caaaaggcca
     4441 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4501 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4561 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4621 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4681 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4741 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4801 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4861 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4921 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4981 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5041 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggatt atcaaaaagg
     5101 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5161 gagtaaactt ggtctgacag ttacc
//
