LOCUS       sequence-364990-        6863 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' 
            Tactb insulator sequence (Tactb-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA). 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6863)
  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 6863)
  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: pTS2304-Tier1-Tactb-PPGK-NLuc_TB
FEATURES             Location/Qualifiers
     source          1..6863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..94)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(248..836)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(328..347)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1007..1798,1799..1867))
                     /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     1630..1649
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1868..1972)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2450..2949
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2485..2508)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2882..2901
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     3002..3058
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             3080..3592
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             3620..3676
                     /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             3695..4402
                     /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(4457..4474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4463..4687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4733..5161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4820..4839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5030..5051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5170..5190)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5175..5504
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5355..5490
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5417..5436
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5571..6365
                     /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(5625..5644)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6235..6254
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6539..6660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6576..6595)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6630..6649
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6709..6725)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6709..6725)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6722..6744)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6733..6749
                     /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(6757..6763,6764..6781,6782..6787))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6802..6823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
       61 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      121 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      181 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      241 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
      301 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
      361 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
      421 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
      481 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
      541 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
      601 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
      661 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
      721 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
      781 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
      841 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
      901 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
      961 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1021 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1081 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1141 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1201 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1261 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1321 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1381 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1441 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1501 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     1561 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     1621 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     1681 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     1741 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     1801 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     1861 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     1921 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     1981 cccgaaaagt gccacctgac gtcgacggat cgggagatct ccacgcgtgg tggggcagtg
     2041 ggggccaact tgtccttacc cagagtgcag gtgtgtggag atccctcctg ccttgacatt
     2101 gagcagcctt agagggtggg ggaggctcag gggtcaggtc tctgttcctg cttattgggg
     2161 agttcctggc ctggcccttc tatgtctccc caggtacccc agtttttctg ggttcaccca
     2221 gagtgcagat gcttgaggag gtgggaaggg actatttggg ggtgtctggc tcaggtgcca
     2281 tgcctcactg gggctggttg gcacctgcat ttcctgggag tggggctgtc tcagggtagc
     2341 tgggcacggt gttcccttga gtgggggtgt agtgggtgtt cctagctgcc acgcctttgc
     2401 cttcacctat gggatcgtgg ctgtcagaac caggtaccct cgagctaccg ggtaggggag
     2461 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     2521 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     2581 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     2641 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcagtagt
     2701 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     2761 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     2821 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     2881 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     2941 ggcctttcga cctccagccc gagctcggta taggttcgaa gcggaattca ccatgactag
     3001 tgagacagac acactcctgc tatgggtact gctgctctgg gttccaggtt ccactggtga
     3061 cgctagtggt ggttctggta tggtcttcac actcgaagat ttcgttgggg actggcgaca
     3121 gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca
     3181 gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct
     3241 gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca
     3301 gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct
     3361 gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg
     3421 gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg
     3481 gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg
     3541 agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cggctagtgg
     3601 tggttctggt ggaagcggag ctactaactt cagcctgctg aagcaggctg gtgacgtgga
     3661 ggagaaccct ggacctgcta gtggtggttc tggtgtgtct aagggcgaag agctgattaa
     3721 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     3781 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     3841 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     3901 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     3961 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     4021 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     4081 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
     4141 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     4201 cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac ccgctaagaa
     4261 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     4321 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     4381 caaactgggg cacaaactta atgctagcgg atccaccggt gtctagaaag ctttgaggcc
     4441 ggcctgcagg gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4501 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4561 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4621 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4681 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4741 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4801 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4861 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4921 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4981 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5041 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5101 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5161 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5221 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5281 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5341 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5401 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     5461 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     5521 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     5581 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     5641 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     5701 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     5761 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     5821 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5881 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5941 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     6001 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     6061 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     6121 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     6181 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     6241 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     6301 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     6361 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     6421 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     6481 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6541 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6601 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6661 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     6721 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6781 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6841 gcccgctttc cagtcgggaa acc
//
