LOCUS       sequence-365617-        4668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PPGK-driven secreted Nluc coupled to 
            fluorescent reporter mTagBFP2 
            (A1-pA::PPGK-IgkSS-Nluc-p2A-mTagBFP2-pA::A3-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4668)
  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 4668)
  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: pTS1040
FEATURES             Location/Qualifiers
     source          1..4668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(63..854,855..923))
                     /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     686..705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(924..1028)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1189..1206)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1195..1419
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1467..1966
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1502..1525)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1899..1918
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     2019..2075
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             2097..2609
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2637..2693
                     /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             2712..3419
                     /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     complement(3541..3557)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3541..3557)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3713..3734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3851..3868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4022..4610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4102..4121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
       61 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      121 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      181 cccagtgctg caatgatacc gcggctccca cgctcaccgg ctccagattt atcagcaata
      241 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      301 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      361 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      421 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      481 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
      541 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
      601 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
      661 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
      721 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
      781 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
      841 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
      901 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
      961 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1021 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccaa gatctccacg
     1081 cgtggtaccc tcgaggagct cttcgaagcg gaattcacca tgactagtgc tagcggatcc
     1141 accggtgtct agaaagcttt gaggccggcc tgcaggcccg ctgatcagcc tcgactgtgc
     1201 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1261 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1321 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1381 acaatagcag gcatgctggg gatgcggtgg gctctatggt agcttaggag gcggaaagaa
     1441 ccagcgcgtg gtaccctcga gctaccgggt aggggaggcg cttttcccaa ggcagtctgg
     1501 agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg
     1561 cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc
     1621 gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
     1681 caggacgtga caaatggaag tagcacgtct cagtagtctc gtgcagatgg acagcaccgc
     1741 tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc
     1801 tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
     1861 gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg
     1921 cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct ccagcccgag
     1981 ctcggtatag gttcgaagcg gaattcacca tgactagtga gacagacaca ctcctgctat
     2041 gggtactgct gctctgggtt ccaggttcca ctggtgacgc tagtggtggt tctggtatgg
     2101 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
     2161 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
     2221 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
     2281 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     2341 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     2401 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     2461 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     2521 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     2581 gctggcggct gtgcgaacgc attctggcgg ctagtggtgg ttctggtgga agcggagcta
     2641 ctaacttcag cctgctgaag caggctggtg acgtggagga gaaccctgga cctgctagtg
     2701 gtggttctgg tgtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt
     2761 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc
     2821 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     2881 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc
     2941 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca
     3001 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     3061 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc
     3121 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc
     3181 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg
     3241 ccaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact
     3301 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc
     3361 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatg
     3421 ctagcggatc caccggtgtc tagaaagcta tacaggtgca ccgctttata ataaaactga
     3481 tacagagaac gttatcatgt gtgttggttt tttttgtgtg tgcggtttaa gggagcctcg
     3541 gtcatagctg tttcctgtgt aaaacgacgg ccagcgcggg agacggtcag ttcgtctcca
     3601 gctcagaaat tatgagagcg tttaataaag tattgactat ggttgacaaa acgtgtgttg
     3661 gagttttttg tgtgctttgt catcaactga gccagtaggt cgagggtgcc taatgagtga
     3721 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4021 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4081 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4201 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4441 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4621 tgatcttttc tacggggatt atcaaaaagg atcttcacct agatcctt
//
