LOCUS       sequence-365630-        4962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable stable integration of up to three cassettes
            via Piggy Bac transposase including a mTagBFP2 marker and BlastR 
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-mTagBFP2-p2A-BlastR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4962)
  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 4962)
  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: pTS2357_Tier3(PB)-TagBFP_BlastR
FEATURES             Location/Qualifiers
     source          1..4962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   264..326
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        386..490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(491..559,560..1351)
                     /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     complement(709..728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1472..2060
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1961..1980
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2132..2166
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2496..2517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3059..3115)
                     /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             complement(3143..3850)
                     /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"
     primer_bind     4540..4556
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4540..4556
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4702..4926
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4915..4932
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 agctttctag acaccggtgg atccgctagc actagtcatg gtgaattccg cttcgaagag
       61 ctcctcgagg gtaccacgcg tagatctctt aaggttttgt tactttatag aagaaatttt
      121 gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
      181 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
      241 tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac
      301 gtcacaatat gattatcttt ctagggttaa gcggccgctg atcttggttt cttagacgtc
      361 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
      421 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      481 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      541 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      601 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      661 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      721 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      781 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      841 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      901 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      961 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1021 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1081 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1141 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     1201 gcgtgggagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1261 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1321 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1381 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1441 taatccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1501 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1561 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1621 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1681 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1741 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1801 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1861 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1921 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1981 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2041 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2101 gctggccttt tgctcacatg tccatggtta accctagaaa gatagtctgc gtaaaattga
     2161 cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg tcgctgtgca
     2221 tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca atgcggtaag
     2281 tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta
     2341 cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt tctacttacg
     2401 tgataactta ttatatatat attttcttgt tatagatatc gcagctggca cgacaggttt
     2461 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     2521 gcaccctcga cctactggct cagttgatga caaagcacac aaaaaactcc aacacacgtt
     2581 ttgtcaacca tagtcaatac tttattaaac gctctcataa tttctgagct ttctagacac
     2641 cggtggatcc gctagcgccc tcccacacat aaccagaggg cagcaattca cgaatcccaa
     2701 ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga
     2761 gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg
     2821 cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt
     2881 ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc
     2941 tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag
     3001 ccgttgctct ttcaatgagg gtggattctt cttgagacaa accagaacca ccactagcag
     3061 gtccagggtt ctcctccacg tcaccagcct gcttcagcag gctgaagtta gtagctccgc
     3121 ttccaccaga accaccacta gcattaagtt tgtgccccag tttgctaggg aggtcgcagt
     3181 atctggccac tgccacctcg tgctgctcga cgtaggtttc gttgttggcc tccttgattc
     3241 tttccagtct gtagtccaca tagtagacgc caggcatctt gaggttctta gcgggtttct
     3301 tggatctata tgtggtcttg gcgtttgcga tcagatggct cccgcccacg agcttcaggg
     3361 ccatgtcgtt tctgccttcc aggccgccgt cagcggggta cagcgtctcg gtgaaggcct
     3421 cccagccgag tgttttcttc tgcatcacag ggccgttgga tgtgaagttc acccctctga
     3481 tcttgacgtt gtagatgagg cagccgtcct ggaggctggt gtcctgggta gcggtcagca
     3541 cgcccccgtc ttcgtatgtg gtgactctct cccatgtgaa gccctcaggg aaggactgct
     3601 tgaagaagtc ggggatgccc tgggtgtggt tgatgaaggt cttgctgccg tagaggaagc
     3661 tagtagccag gatgtcgaag gcgaagggga gagggccgcc ctcgaccacc ttgattctca
     3721 tggtctgggt gccctcgtag ggcttgcctt cgccctcgga tgtgcacttg aagtgatggt
     3781 tgtccacggt gccctccatg tacagcttca tgtgcatgtt ctccttaatc agctcttcgc
     3841 ccttagacac accagaacca ccactagtca tggtgaattc gagctcccta ggtgcagagg
     3901 tttctacagg gaaagagaga aaggctacag ttagtttgct gtgatgatgc taagtgccga
     3961 ggtcagatgt ctatggattg tacacggaat cccaacgcag gaaccaaaaa acactcaccc
     4021 aaaaaaatgg cgctaaggcc gagagaaagg cgaagttccg tctacggcta tataccttga
     4081 aacaggggcg gcagaagtga cgcaacgcgg ttgcgcggac ggcttcctgt gttttcattg
     4141 gtcgaagcgt tctgcgcgtg cgcagagatt ccccagggaa ggcgggccct cttccgaatg
     4201 tccaatccgg acattcttcc ggttggacct ccgcggtcag cgctgtgccc ggcgagagat
     4261 cacgtggggc gcggaggcgg tgctgctggg gcacggccgt ccagcctcgg cggccatatt
     4321 tttgaggggc tgttcatctc gttcacacgc tctgtccgcc atgtttgtga gtggaagcgc
     4381 cattacccct tcaagcgact gaaggctgca gggcctctgg tggcccgcat ggggagtcca
     4441 gacccgccag gcccgccttt ccgcactcag tccgggctta ctttattttg tgagacaggg
     4501 tcacgctcga gggtaccacg cgctggccgt cgttttacac aggaaacagc tatgaccgag
     4561 gctcccttaa accgcacaca caaaaaaaac caacacacat gataacgttc tctgtatcag
     4621 ttttattata aagcggtgca cctgtatagc tggagaccaa ctgacggtct cccgcgctgg
     4681 ttctttccgc ctcctaagct accatagagc ccaccgcatc cccagcatgc ctgctattgt
     4741 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
     4801 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
     4861 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
     4921 gcacagtcga ggctgatcag cgggcctgca ggccggcctc aa
//
