LOCUS       sequence-365612-        8339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 co-expression vector encoding PhCMV-driven SEAP coupled to 
            fluorescent reporter iRFP670 and PmPGK1-driven secreted Nluc coupled
            to fluorescent reporter mTagBFP2 that is insulated via a 5' CoTC 
            insulator sequence 
            (PhCMV-SEAP-p2A-iRFP670-pA::A2-pA::CoTC-PmPGK1-IgkSS-Nluc-p2A-mTagBF
            P2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8339)
  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 8339)
  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: pTS2312
FEATURES             Location/Qualifiers
     source          1..8339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(75..866,867..935))
                     /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     698..717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(936..1040)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1110..1489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1490..1693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1644..1664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1738..1757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1738..1756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3375..3431
                     /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             3438..4367
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     primer_bind     complement(4428..4445)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4434..4658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4804..4820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5304..5803
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5339..5362)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5736..5755
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5856..5912
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5934..6446
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6474..6530
                     /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             6549..7256
                     /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    7396..7417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7534..7551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7705..8293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7785..7804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
       61 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      121 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
      181 ttaccatctg gccccagtgc tgcaatgata ccgcggctcc cacgctcacc ggctccagat
      241 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
      301 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
      361 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
      421 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
      481 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
      541 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
      601 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
      661 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
      721 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
      781 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
      841 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
      901 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
      961 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1021 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     1081 aagatctcca cgcgtggtac cctcgagttg acattgatta ttgagtagtt attaatagta
     1141 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     1201 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     1261 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     1321 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     1381 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     1441 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     1501 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1561 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1621 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1681 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     1741 tacgactcac tatagggaga cccaagctgt tcgaagcgga attcaccatg actagtctgc
     1801 tgctgctgct gctgggcctg agactgcagc tgagcctggg catcatcccc gtggaggagg
     1861 agaaccccga cttctggaac agagaggccg ccgaggccct gggcgccgcc aagaagctgc
     1921 agcccgccca gaccgccgcc aagaacctga tcatcttcct gggcgacggc atgggcgtga
     1981 gcaccgtgac cgccgccaga atcctgaagg gccagaagaa ggacaagctg ggccccgaga
     2041 tccccctggc catggacaga ttcccctacg tggccctgag caagacctac aacgtggaca
     2101 agcacgtgcc cgacagcggc gccaccgcca ccgcctacct gtgcggcgtg aagggcaact
     2161 tccagaccat cggcctgagc gccgccgcca gattcaacca gtgcaacacc accagaggca
     2221 acgaggtgat cagcgtgatg aacagagcca agaaggccgg caagagcgtg ggcgtggtga
     2281 ccaccaccag agtgcagcac gccagccccg ccggcaccta cgcccacacc gtgaacagaa
     2341 actggtacag cgacgccgac gtgcccgcca gcgccagaca ggagggctgc caggacatcg
     2401 ccacccagct gatcagcaac atggacatcg acgtgatcct gggcggcggc agaaagtaca
     2461 tgttcagaat gggcaccccc gaccccgagt accccgacga ctacagccag ggcggcacca
     2521 gactggacgg caagaacctg gtgcaggagt ggctggccaa gagacagggc gccagatacg
     2581 tgtggaacag aaccgagctg atgcaggcca gcctggaccc cagcgtgacc cacctgatgg
     2641 gcctgttcga gcccggcgac atgaagtacg agatccacag agacagcacc ctggacccca
     2701 gcctgatgga gatgaccgag gccgccctga gactgctgag cagaaacccc agaggcttct
     2761 tcctgttcgt ggagggcggc agaatcgacc acggccacca cgagagcaga gcctacagag
     2821 ccctgaccga gaccatcatg ttcgacgacg ccatcgagag agccggccag ctgaccagcg
     2881 aggaggacac cctgagcctg gtgaccgccg accacagcca cgtgttcagc ttcggcggct
     2941 accccctgag aggcagcagc atcttcggcc tggcccccgg caaggccaga gacagaaagg
     3001 cctacaccgt gctgctgtac ggcaacggcc ccggctacgt gctgaaggac ggcgccagac
     3061 ccgacgtgac cgagagcgag agcggcagcc ccgagtacag acagcagagc gccgtgcccc
     3121 tggacgagga gacccacgcc ggcgaggacg tggccgtgtt cgccagaggc ccccaggccc
     3181 acctggtgca cggcgtgcag gagcagacct tcatcgccca cgtgatggcc ttcgccgcct
     3241 gcctggagcc ctacaccgcc tgcgacctgg ccccccccgc cggcaccacc gacgccgccc
     3301 accccggcta cagcagagtg ggcgccgccg gcagattcga gcagaccgct agtggtggtt
     3361 ctggtggaag cggagctact aacttcagcc tgctgaagca ggctggtgac gtggaggaga
     3421 accctggacc tgctagtgcg cgtaaggtcg atctcacctc ctgcgatcgc gagccgatcc
     3481 acatccccgg cagcattcag ccgtgcggct gcctgcttgc ctgcgacgcg caggcggtgc
     3541 ggatcacgcg cattacggaa aatgccggcg cgttctttgg acgcgaaact ccgcgggtcg
     3601 gtgagctact cgccgattac ttcggcgaga ccgaagccca tgcgctgcgc aacgcactgg
     3661 cgcagtcctc cgatccaaag cgaccggcgc tgatcttcgg ttggcgcgac ggcctgaccg
     3721 gccgcacctt cgacatctca ctgcatcgcc atgacggtac atcgatcatc gagttcgagc
     3781 ctgcggcggc cgaacaggcc gacaatccgc tgcggctgac gcggcagatc atcgcgcgca
     3841 ccaaagaact gaagtcgctc gaagagatgg ccgcacgggt gccgcgctat ctgcaggcga
     3901 tgctcggcta tcaccgcgtg atgttgtacc gcttcgcgga cgacggctcc gggatggtga
     3961 tcggcgaggc gaagcgcagc gacctcgaga gctttctcgg tcagcacttt ccggcgtcgc
     4021 tggtcccgca gcaggcgcgg ctactgtact tgaagaacgc gatccgcgtg gtctcggatt
     4081 cgcgcggcat cagcagccgg atcgtgcccg agcacgacgc ctccggcgcc gcgctcgatc
     4141 tgtcgttcgc gcacctgcgc agcatctcgc cctgccatct cgaatttctg cggaacatgg
     4201 gcgtcagcgc ctcgatgtcg ctgtcgatca tcattgacgg cacgctatgg ggattgatca
     4261 tctgtcatca ttacgagccg cgtgccgtgc cgatggcgca gcgcgtcgcg gccgaaatgt
     4321 tcgccgactt cttatcgctg cacttcaccg ccgcccacca ccaacgcgct agcggatcca
     4381 ccggtgtcta gaaagctttg aggccggcct gcaggcccgc tgatcagcct cgactgtgcc
     4441 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4501 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4561 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4621 caatagcagg catgctgggg atgcggtggg ctctatggta gcttaggagg cggaaagaac
     4681 cagcgcggga gaccgtcagt tggtctccag ctatacaggt gcaccgcttt ataataaaac
     4741 tgatacagag aacgttatca tgtgtgttgg ttttttttgt gtgtgcggtt taagggagcc
     4801 tcggtcatag ctgtttcctg tgtaaaacga cggccagcgc gtatgtccct tatggtgctt
     4861 ctggctctgc agttattagc atagtgttac catcaaccac cttaacttca tttttcttat
     4921 tcaataccta ggtaggtaga tgctagattc tggaaataaa atatgagtct caagtggtcc
     4981 ttgtcctctc tcccagtcaa attctgaatc tagttggcaa gattctgaaa tcaaggcata
     5041 taatcagtaa taagtgatga tagaagggta tatagaagaa ttttattata tgagagggtg
     5101 aaaccctcaa aatgaaatga aatcagaccc ttgtcttaca ccataaacaa aaataaattt
     5161 gaatgggtta aagaattaaa ctaagaccta aaaccataaa aatttttaaa gaaatcaaaa
     5221 gaagaaaatt ctaatattca cgttgcagcc gttttttgaa tttgatatga gaagcaaagg
     5281 caacaaggta ccctcgagct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     5341 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     5401 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     5461 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     5521 gacgtgacaa atggaagtag cacgtctcag tagtctcgtg cagatggaca gcaccgctga
     5581 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     5641 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     5701 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     5761 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctcca gcccgagctc
     5821 ggtataggtt cgaagcggaa ttcaccatga ctagtgagac agacacactc ctgctatggg
     5881 tactgctgct ctgggttcca ggttccactg gtgacgctag tggtggttct ggtatggtct
     5941 tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac ctggaccaag
     6001 tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc gtaactccga
     6061 tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc
     6121 cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt aaggtggtgt
     6181 accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg gtaatcgacg
     6241 gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc gccgtgttcg
     6301 acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt atcgacgagc
     6361 gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga gtgaccggct
     6421 ggcggctgtg cgaacgcatt ctggcggcta gtggtggttc tggtggaagc ggagctacta
     6481 acttcagcct gctgaagcag gctggtgacg tggaggagaa ccctggacct gctagtggtg
     6541 gttctggtgt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
     6601 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     6661 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     6721 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     6781 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     6841 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     6901 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     6961 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     7021 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca
     7081 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     7141 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     7201 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaatgcta
     7261 gcggatccac cggtgtctag aaagctcaga aattatgaga gcgtttaata aagtattgac
     7321 tatggttgac aaaacgtgtg ttggagtttt ttgtgtgctt tgtcatcaac tgagccagta
     7381 ggtcgagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     7441 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     7501 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     7561 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     7621 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     7681 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     7741 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7801 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     7861 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     7921 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     7981 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     8041 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     8101 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     8161 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     8221 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     8281 aaaaggatct caagaagatc ctttgatctt ttctacgggg attatcaaaa aggatcttc
//
