LOCUS       sequence-366213-        5979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven delayed-maturing fluorescent 
            protein fluorescent timer (FT) with ""fast"" maturation fastFT fused
            to a ""self-cleaving"" peptide p2a (PhCMV-p2a_medFT-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5979)
  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 5979)
  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: pTS2393-Tier1-PhCMV-p2a_fastFT
FEATURES             Location/Qualifiers
     source          1..5979
                     /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
     enhancer        1945..2324
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2325..2528
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2479..2499
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2573..2592
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2573..2591
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2653..2709
                     /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             2716..3420
                     /codon_start=1
                     /product="blue-to-red fluorescent timer derivative of 
                     mCherry (Subach et al., 2009)"
                     /label=Fast-FT
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHVEGSVNGHEFEIEGEGKGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSRAYVKHPADIPDYWKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERSE
                     GRHSTGGMDELYK"
     primer_bind     complement(2858..2876)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3126..3145
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3475..3492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3481..3705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3751..4179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3838..3857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4048..4069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4188..4208)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4193..4522
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4373..4508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4435..4454
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4589..5383
                     /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(4643..4662)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5253..5272
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5557..5678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5594..5613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5648..5667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5727..5743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5727..5743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5740..5762)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5751..5767
                     /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(5775..5781,5782..5799,5800..5805))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5820..5841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5958..5975)
                     /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 ggtaccctcg agttgacatt gattattgag tagttattaa tagtaatcaa
     1981 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2041 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2101 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2161 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2221 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2281 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2341 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2401 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2461 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2521 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     2581 ctcactatag ggagacccaa gctgttcgaa gcggaattca ccatgactag tggtggttct
     2641 ggtggaagcg gagctactaa cttcagcctg ctgaagcagg ctggtgacgt ggaggagaac
     2701 cctggacctg ctagtgtgag caagggcgag gaggataaca tggccatcat caaagagttc
     2761 atgcgtttca aagtgcacgt ggagggttcc gtgaacggcc acgagttcga gatcgagggc
     2821 gagggcaagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     2881 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc
     2941 tacgtgaagc atcccgccga catccccgac tactggaagc tgtccttccc cgagggcttc
     3001 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     3061 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggaactaa cttcccctcc
     3121 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga gcggatgtac
     3181 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     3241 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     3301 gcttacaacg tcaatatcaa gttggacatc acatcccaca acgaggacta caccatcgtg
     3361 gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     3421 gctagcggat ccaccggtgt ctagaaagct ttgaggccgg cctgcaggga tcagcctcga
     3481 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3541 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3601 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3661 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3721 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3781 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3841 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3901 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3961 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4021 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4081 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4141 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4201 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4261 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4321 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4381 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4441 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     4501 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     4561 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4621 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4681 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4741 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4801 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4861 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4921 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4981 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5041 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5101 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5161 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5221 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5281 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5341 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     5401 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5461 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5521 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5581 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5641 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5701 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5761 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5821 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5881 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5941 gggcgctctt ccgcttcctc gctcactgac tcgctgcgc
//
