LOCUS       sequence-365633-        5382 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 iRFP670 marker and PuroR 
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-iRFP670-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5382)
  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 5382)
  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: pTS2355_Tier3(PB)-iRFP_PuroR
FEATURES             Location/Qualifiers
     source          1..5382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(28..819,820..888))
                     /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     651..670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(889..993)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   1053..1115
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1409..1426)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1415..1639
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1785..1801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1785..1801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2479..3408
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             3436..3492
                     /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             3511..4104
                     /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     4004..4024
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4244..4265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4595..4629
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(4701..5289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4781..4800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
       61 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
      121 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggc tcccacgctc
      181 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
      241 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
      301 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
      361 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
      421 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
      481 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
      541 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
      601 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
      661 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
      721 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
      781 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
      841 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
      901 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
      961 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     1021 cgtctaagaa accaagatca gcggccgctt aaccctagaa agataatcat attgtgacgt
     1081 acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg tatatcgagg
     1141 tttatttatt aatttgaata gatattaagt tttattatat ttacacttac atactaataa
     1201 taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaac aaaaactcaa
     1261 aatttcttct ataaagtaac aaaaccttaa gagatctacg cgtggtaccc tcgaggagct
     1321 cttcgaagcg gaattcacca tgactagtgc tagcggatcc accggtgtct agaaagcttt
     1381 gaggccggcc tgcaggcccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1441 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1501 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1561 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1621 gatgcggtgg gctctatggt agcttaggag gcggaaagaa ccagcgcggg agaccgtcag
     1681 ttggtctcca gctatacagg tgcaccgctt tataataaaa ctgatacaga gaacgttatc
     1741 atgtgtgttg gttttttttg tgtgtgcggt ttaagggagc ctcggtcata gctgtttcct
     1801 gtgtaaaacg acggccagcg cgtggtaccc tcgagcgtga ccctgtctca caaaataaag
     1861 taagcccgga ctgagtgcgg aaaggcgggc ctggcgggtc tggactcccc atgcgggcca
     1921 ccagaggccc tgcagccttc agtcgcttga aggggtaatg gcgcttccac tcacaaacat
     1981 ggcggacaga gcgtgtgaac gagatgaaca gcccctcaaa aatatggccg ccgaggctgg
     2041 acggccgtgc cccagcagca ccgcctccgc gccccacgtg atctctcgcc gggcacagcg
     2101 ctgaccgcgg aggtccaacc ggaagaatgt ccggattgga cattcggaag agggcccgcc
     2161 ttccctgggg aatctctgcg cacgcgcaga acgcttcgac caatgaaaac acaggaagcc
     2221 gtccgcgcaa ccgcgttgcg tcacttctgc cgcccctgtt tcaaggtata tagccgtaga
     2281 cggaacttcg cctttctctc ggccttagcg ccattttttt gggtgagtgt tttttggttc
     2341 ctgcgttggg attccgtgta caatccatag acatctgacc tcggcactta gcatcatcac
     2401 agcaaactaa ctgtagcctt tctctctttc cctgtagaaa cctctgcacc tagggagctc
     2461 gaattcacca tgactagtgc gcgtaaggtc gatctcacct cctgcgatcg cgagccgatc
     2521 cacatccccg gcagcattca gccgtgcggc tgcctgcttg cctgcgacgc gcaggcggtg
     2581 cggatcacgc gcattacgga aaatgccggc gcgttctttg gacgcgaaac tccgcgggtc
     2641 ggtgagctac tcgccgatta cttcggcgag accgaagccc atgcgctgcg caacgcactg
     2701 gcgcagtcct ccgatccaaa gcgaccggcg ctgatcttcg gttggcgcga cggcctgacc
     2761 ggccgcacct tcgacatctc actgcatcgc catgacggta catcgatcat cgagttcgag
     2821 cctgcggcgg ccgaacaggc cgacaatccg ctgcggctga cgcggcagat catcgcgcgc
     2881 accaaagaac tgaagtcgct cgaagagatg gccgcacggg tgccgcgcta tctgcaggcg
     2941 atgctcggct atcaccgcgt gatgttgtac cgcttcgcgg acgacggctc cgggatggtg
     3001 atcggcgagg cgaagcgcag cgacctcgag agctttctcg gtcagcactt tccggcgtcg
     3061 ctggtcccgc agcaggcgcg gctactgtac ttgaagaacg cgatccgcgt ggtctcggat
     3121 tcgcgcggca tcagcagccg gatcgtgccc gagcacgacg cctccggcgc cgcgctcgat
     3181 ctgtcgttcg cgcacctgcg cagcatctcg ccctgccatc tcgaatttct gcggaacatg
     3241 ggcgtcagcg cctcgatgtc gctgtcgatc atcattgacg gcacgctatg gggattgatc
     3301 atctgtcatc attacgagcc gcgtgccgtg ccgatggcgc agcgcgtcgc ggccgaaatg
     3361 ttcgccgact tcttatcgct gcacttcacc gccgcccacc accaacgcgc tagtggtggt
     3421 tctggtggaa gcggagctac taacttcagc ctgctgaagc aggctggtga cgtggaggag
     3481 aaccctggac ctgctagtgg tggttctggt accgagtaca agcccacggt gcgcctcgcc
     3541 acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     3601 gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3661 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3721 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3781 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3841 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtgtcg
     3901 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3961 gagcgcgccg gggtgcccgc cttcctggag acatccgcgc cccgcaacct ccccttctac
     4021 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     4081 tgcatgaccc gcaagcccgg tgccgctagc ggatccaccg gtgtctagaa agctcagaaa
     4141 ttatgagagc gtttaataaa gtattgacta tggttgacaa aacgtgtgtt ggagtttttt
     4201 gtgtgctttg tcatcaactg agccagtagg tcgagggtgc ctaatgagtg agctaactca
     4261 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4321 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
     4381 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
     4441 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
     4501 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
     4561 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
     4621 tttctagggt taaccatgga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4681 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4741 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4801 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4861 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4921 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4981 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5041 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5101 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5161 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5221 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5281 ggatctcaag aagatccttt gatcttttct acggggatta tcaaaaagga tcttcaccta
     5341 gatcctttta aattaaaaat gaagttttaa atcaatctaa ag
//
