LOCUS       sequence-364981-        6613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 expression vector encoding PmPGK1-driven secreted Nluc 
            coupled to fluorescent reporter mTagBFP2 that is insulated via a 5' 
            insulator sequence derived from the pGL3 plasmid 
            (pGL3-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6613)
  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 6613)
  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: pTS2308-Tier1-pGL3-PPGK-NLuc_TB
FEATURES             Location/Qualifiers
     source          1..6613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(9..26)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(180..768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(260..279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(939..1730,1731..1799))
                     /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     1562..1581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1800..1904)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    1961..2009
                     /note="synthetic polyadenylation signal"
     misc_feature    2023..2114
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2063..2082
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        2132..2631
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2167..2190)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2564..2583
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     2684..2740
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             2762..3274
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             3302..3358
                     /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             3377..4084
                     /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(4139..4156)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4145..4369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4415..4843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4502..4521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4712..4733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4852..4872)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4857..5186
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5037..5172
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5099..5118
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5253..6047
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5307..5326)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5917..5936
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6221..6342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6258..6277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6312..6331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6391..6407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6391..6407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6404..6426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6415..6431
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6439..6445,6446..6463,6464..6469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6484..6505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
       61 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      121 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      181 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      241 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      301 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      361 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      421 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      481 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      541 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      601 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
      661 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      721 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
      781 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
      841 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
      901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
      961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1081 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1141 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1201 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1261 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1321 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1381 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1441 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1501 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     1561 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     1621 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     1681 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     1741 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     1801 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     1861 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     1921 gtgccacctg acgtcgacgg atcgggagat ctccacgcgt aataaaatat ctttattttc
     1981 attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg ctctccatca
     2041 aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc
     2101 cagaacattt ctctggtacc ctcgagctac cgggtagggg aggcgctttt cccaaggcag
     2161 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     2221 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     2281 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     2341 tcgtgcagga cgtgacaaat ggaagtagca cgtctcagta gtctcgtgca gatggacagc
     2401 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     2461 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     2521 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     2581 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctccagc
     2641 ccgagctcgg tataggttcg aagcggaatt caccatgact agtgagacag acacactcct
     2701 gctatgggta ctgctgctct gggttccagg ttccactggt gacgctagtg gtggttctgg
     2761 tatggtcttc acactcgaag atttcgttgg ggactggcga cagacagccg gctacaacct
     2821 ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt cagaatctcg gggtgtccgt
     2881 aactccgatc caaaggattg tcctgagcgg tgaaaatggg ctgaagatcg acatccatgt
     2941 catcatcccg tatgaaggtc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa
     3001 ggtggtgtac cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt
     3061 aatcgacggg gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc
     3121 cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat
     3181 cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt
     3241 gaccggctgg cggctgtgcg aacgcattct ggcggctagt ggtggttctg gtggaagcgg
     3301 agctactaac ttcagcctgc tgaagcaggc tggtgacgtg gaggagaacc ctggacctgc
     3361 tagtggtggt tctggtgtgt ctaagggcga agagctgatt aaggagaaca tgcacatgaa
     3421 gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg
     3481 caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc
     3541 cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca
     3601 cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca catgggagag
     3661 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     3721 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt
     3781 gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg
     3841 cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc
     3901 aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt
     3961 ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga
     4021 gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaaact
     4081 taatgctagc ggatccaccg gtgtctagaa agctttgagg ccggcctgca gggatcagcc
     4141 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4201 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4261 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4321 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     4381 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     4441 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4501 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4561 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4621 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4681 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4741 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4801 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4861 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4921 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4981 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     5041 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5101 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     5161 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     5221 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     5281 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     5341 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     5401 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     5461 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     5521 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     5581 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     5641 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     5701 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     5761 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     5821 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5881 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5941 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     6001 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     6061 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     6121 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     6181 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     6241 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     6301 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     6361 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6421 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6481 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6541 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6601 tattgggcgc tct
//
