LOCUS       sequence-366634-        4668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PPGK-driven secreted Nluc coupled to 
            fluorescent reporter mTagBFP2 
            (A1-pA::A2-pA::PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-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: pTS1041
FEATURES             Location/Qualifiers
     source          1..4668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(49..840,841..909))
                     /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     672..691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(910..1014)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1175..1192)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1181..1405
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1551..1567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1551..1567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1607..2106
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1642..1665)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2039..2058
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     2159..2215
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             2237..2749
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2777..2833
                     /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             2852..3559
                     /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"
     protein_bind    3699..3720
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3837..3854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4008..4596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4088..4107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
       61 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      121 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      181 gataccgcgg ctcccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      241 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      301 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      361 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      421 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      481 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      541 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      601 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      661 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      721 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      781 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      841 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      901 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      961 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1021 tccccgaaaa gtgccacctg acgtctaaga aaccaagatc tccacgcgtg gtaccctcga
     1081 ggagctcttc gaagcggaat tcaccatgac tagtgctagc ggatccaccg gtgtctagaa
     1141 agctttgagg ccggcctgca ggcccgctga tcagcctcga ctgtgccttc tagttgccag
     1201 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1261 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1321 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1381 gctggggatg cggtgggctc tatggtagct taggaggcgg aaagaaccag cgcgggagac
     1441 cgtcagttgg tctccagcta tacaggtgca ccgctttata ataaaactga tacagagaac
     1501 gttatcatgt gtgttggttt tttttgtgtg tgcggtttaa gggagcctcg gtcatagctg
     1561 tttcctgtgt aaaacgacgg ccagcgcgtg gtaccctcga gctaccgggt aggggaggcg
     1621 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     1681 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
     1741 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     1801 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cagtagtctc
     1861 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     1921 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     1981 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     2041 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     2101 ctttcgacct ccagcccgag ctcggtatag gttcgaagcg gaattcacca tgactagtga
     2161 gacagacaca ctcctgctat gggtactgct gctctgggtt ccaggttcca ctggtgacgc
     2221 tagtggtggt tctggtatgg tcttcacact cgaagatttc gttggggact ggcgacagac
     2281 agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa
     2341 tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa
     2401 gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat
     2461 cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca
     2521 ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc
     2581 gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa
     2641 cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt
     2701 aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgg ctagtggtgg
     2761 ttctggtgga agcggagcta ctaacttcag cctgctgaag caggctggtg acgtggagga
     2821 gaaccctgga cctgctagtg gtggttctgg tgtgtctaag ggcgaagagc tgattaagga
     2881 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
     2941 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
     3001 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
     3061 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
     3121 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     3181 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     3241 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     3301 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     3361 gagccatctg atcgcaaacg ccaagaccac atatagatcc aagaaacccg ctaagaacct
     3421 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     3481 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     3541 actggggcac aaacttaatg ctagcggatc caccggtgtc tagaaagctc agaaattatg
     3601 agagcgttta ataaagtatt gactatggtt gacaaaacgt gtgttggagt tttttgtgtg
     3661 ctttgtcatc aactgagcca gtaggtcgag ggtgcctaat gagtgagcta actcacatta
     3721 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3781 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3841 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3901 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3961 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4021 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4081 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4141 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4201 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4261 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4321 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4381 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4441 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4501 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4561 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4621 gggattatca aaaaggatct tcacctagat ccttttaaat taaaaatg
//
