LOCUS       sequence-364524-        6903 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' 
            CoTC insulator sequence (CoTC-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  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 6903)
  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: pTS2303-Tier1-CoTC-PPGK-NLuc_TB
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(53..70)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(224..812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(304..323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(983..1774,1775..1843))
                     /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     1606..1625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1844..1948)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2466..2965
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2501..2524)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2898..2917
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     3018..3074
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             3096..3608
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             3636..3692
                     /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             3711..4418
                     /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(4473..4490)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4479..4703
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4749..5177
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4836..4855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5046..5067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5186..5206)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5191..5520
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5371..5506
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5433..5452
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5587..6381
                     /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(5641..5660)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6251..6270
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6555..6676
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6592..6611)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6646..6665
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6725..6741)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6725..6741)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6738..6760)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6749..6765
                     /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(6773..6779,6780..6797,6798..6803))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6818..6839
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
       61 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      121 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      181 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      241 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      301 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      361 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      421 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      481 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      541 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      601 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      661 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      721 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      781 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
      841 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
      901 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
      961 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1021 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1081 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1141 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1201 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1261 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     1321 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1381 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1441 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1501 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1561 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1621 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1681 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1741 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1801 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1861 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1921 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     1981 acggatcggg agatctccac gcgtatgtcc cttatggtgc ttctggctct gcagttatta
     2041 gcatagtgtt accatcaacc accttaactt catttttctt attcaatacc taggtaggta
     2101 gatgctagat tctggaaata aaatatgagt ctcaagtggt ccttgtcctc tctcccagtc
     2161 aaattctgaa tctagttggc aagattctga aatcaaggca tataatcagt aataagtgat
     2221 gatagaaggg tatatagaag aattttatta tatgagaggg tgaaaccctc aaaatgaaat
     2281 gaaatcagac ccttgtctta caccataaac aaaaataaat ttgaatgggt taaagaatta
     2341 aactaagacc taaaaccata aaaattttta aagaaatcaa aagaagaaaa ttctaatatt
     2401 cacgttgcag ccgttttttg aatttgatat gagaagcaaa ggcaacaagg taccctcgag
     2461 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     2521 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     2581 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     2641 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     2701 agcacgtctc agtagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     2761 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     2821 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     2881 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     2941 ctcttcctca tctccgggcc tttcgacctc cagcccgagc tcggtatagg ttcgaagcgg
     3001 aattcaccat gactagtgag acagacacac tcctgctatg ggtactgctg ctctgggttc
     3061 caggttccac tggtgacgct agtggtggtt ctggtatggt cttcacactc gaagatttcg
     3121 ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg
     3181 tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga
     3241 gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg
     3301 gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc
     3361 actttaaggt gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga
     3421 tcgactattt cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg
     3481 taacagggac cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg
     3541 gctccctgct gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca
     3601 ttctggcggc tagtggtggt tctggtggaa gcggagctac taacttcagc ctgctgaagc
     3661 aggctggtga cgtggaggag aaccctggac ctgctagtgg tggttctggt gtgtctaagg
     3721 gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca
     3781 accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     3841 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta
     3901 gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca
     3961 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     4021 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     4081 tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     4141 aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg
     4201 ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca
     4261 agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa
     4321 gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat
     4381 actgcgacct ccctagcaaa ctggggcaca aacttaatgc tagcggatcc accggtgtct
     4441 agaaagcttt gaggccggcc tgcagggatc agcctcgact gtgccttcta gttgccagcc
     4501 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4561 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4621 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4681 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     4741 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4801 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4861 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4921 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4981 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5041 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5101 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5161 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     5221 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     5281 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     5341 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5401 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     5461 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     5521 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     5581 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     5641 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     5701 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     5761 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     5821 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5881 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5941 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     6001 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     6061 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     6121 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     6181 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     6241 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     6301 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     6361 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     6421 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     6481 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     6541 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     6601 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     6661 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     6721 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     6781 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6841 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6901 gaa
//
