LOCUS       sequence-366597-        6935 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 
            tandem repeat of a 5' cHS4 insulator sequence 
            (2xcHS4-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6935)
  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 6935)
  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: pTS2307-Tier1-2xcHS4-PPGK-NLuc_TB
FEATURES             Location/Qualifiers
     source          1..6935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(150..738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(230..249)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(909..1700,1701..1769))
                     /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     1532..1551
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1770..1874)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2424..2923
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2459..2482)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2856..2875
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     2976..3032
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             3054..3566
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             3594..3650
                     /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             3669..4376
                     /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(4431..4448)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4437..4661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4707..5135
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4794..4813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5004..5025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5144..5164)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5149..5478
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5329..5464
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5391..5410
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5545..6339
                     /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(5599..5618)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6209..6228
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6513..6634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6550..6569)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6604..6623
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6683..6699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6683..6699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6696..6718)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6707..6723
                     /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(6731..6737,6738..6755,6756..6761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6776..6797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6914..6931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
       61 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
      121 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
      181 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
      241 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
      301 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
      361 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
      421 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
      481 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
      541 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
      601 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
      661 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
      721 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
      781 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
      841 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
      901 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
      961 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     1021 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     1081 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     1141 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     1201 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     1261 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     1321 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     1381 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     1441 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     1501 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     1561 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     1621 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     1681 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     1741 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     1801 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1861 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     1921 ctccacgcgt ctcgagggga cagccccccc ccaaagcccc cagggatgta attacgtccc
     1981 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
     2041 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
     2101 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
     2161 ggaaaaagct aatcgagggg acagcccccc cccaaagccc ccagggatgt aattacgtcc
     2221 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc
     2281 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga
     2341 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg
     2401 gggaaaaagc taatcgagct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     2461 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     2521 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     2581 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     2641 gacgtgacaa atggaagtag cacgtctcag tagtctcgtg cagatggaca gcaccgctga
     2701 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     2761 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     2821 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     2881 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctcca gcccgagctc
     2941 ggtataggtt cgaagcggaa ttcaccatga ctagtgagac agacacactc ctgctatggg
     3001 tactgctgct ctgggttcca ggttccactg gtgacgctag tggtggttct ggtatggtct
     3061 tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac ctggaccaag
     3121 tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc gtaactccga
     3181 tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc
     3241 cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt aaggtggtgt
     3301 accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg gtaatcgacg
     3361 gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc gccgtgttcg
     3421 acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt atcgacgagc
     3481 gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga gtgaccggct
     3541 ggcggctgtg cgaacgcatt ctggcggcta gtggtggttc tggtggaagc ggagctacta
     3601 acttcagcct gctgaagcag gctggtgacg tggaggagaa ccctggacct gctagtggtg
     3661 gttctggtgt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
     3721 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     3781 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     3841 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     3901 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     3961 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     4021 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     4081 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     4141 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca
     4201 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     4261 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     4321 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaatgcta
     4381 gcggatccac cggtgtctag aaagctttga ggccggcctg cagggatcag cctcgactgt
     4441 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     4501 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     4561 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     4621 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     4681 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     4741 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4801 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4861 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4921 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4981 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5041 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5101 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     5161 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5221 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5281 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     5341 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     5401 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5461 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     5521 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     5581 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     5641 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     5701 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     5761 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     5821 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     5881 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     5941 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     6001 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     6061 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     6121 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     6181 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     6241 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     6301 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     6361 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     6421 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     6481 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     6541 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     6601 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     6661 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6721 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6781 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6841 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6901 gctcttccgc ttcctcgctc actgactcgc tgcgc
//
