LOCUS       sequence-364517-        5895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven delayed-maturing fluorescent 
            protein fluorescent timer (FT) with ""medium"" maturation medFT 
            (PhCMV-medFT-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5895)
  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 5895)
  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: pTS2384-Tier1-PhCMV-medFT
FEATURES             Location/Qualifiers
     source          1..5895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     762..781
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        762..780
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             821..1525
                     /codon_start=1
                     /product="blue-to-red fluorescent timer derivative of 
                     mCherry (Subach et al., 2009)"
                     /label=Medium-FT
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHLEGSVDGHEFEIEGEGEGRPYEGT
                     QSAKLKVTKGGPLPFAWDILSPQFMYGSRAYVKHPADIPDYWKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERIYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVLLPGAYNVNIKMDITSHNEDYTIVEQCERAE
                     GHHSTGGMDELYK"
     primer_bind     complement(963..981)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1231..1250
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1580..1597)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1586..1810
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1856..2284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1943..1962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2153..2174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2293..2313)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2298..2627
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2478..2613
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2540..2559
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2694..3488
                     /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(2748..2767)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3358..3377
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3662..3783
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3699..3718)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3753..3772
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3832..3848)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3832..3848)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3845..3867)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3856..3872
                     /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(3880..3886,3887..3904,3905..3910))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3925..3946
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4063..4080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4234..4822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4314..4333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4993..5784,5785..5853))
                     /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     5616..5635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5854..5895,1..63))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
       61 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tccacgcgtg
      121 gtaccctcga gttgacattg attattgagt agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      721 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      781 gagacccaag ctgttcgaag cggaattcac catgactagt gtgagcaagg gcgaggagga
      841 taacatggcc atcatcaagg agttcatgcg tttcaaggtg cacctggagg gctccgtgga
      901 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagag
      961 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     1021 tcagttcatg tacggctcca gggcctacgt gaagcacccc gccgacatcc ccgactactg
     1081 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggatggcgg
     1141 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     1201 gctgcgcggc accaacttcc cttccgacgg ccccgtaatg cagaagaaga ccatgggctg
     1261 ggaggcctcc tccgagcgga tataccccga ggacggcgcc ctgaagggcg agatcaagca
     1321 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     1381 caagaagccc gtgctgctgc ccggcgccta caacgtcaac atcaagatgg acatcacctc
     1441 ccacaacgag gactacacca tcgttgaaca gtgcgaacgc gccgagggcc accattccac
     1501 cggcggcatg gacgagctgt acaaggctag cggatccacc ggtgtctaga aagctttgag
     1561 gccggcctgc agggatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1621 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1681 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1741 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1801 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     1861 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1921 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1981 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2041 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2101 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2161 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2221 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2281 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2341 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2401 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2461 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2521 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     2581 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     2641 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     2701 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2761 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2821 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     2881 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2941 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3001 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3061 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3121 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3181 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3241 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3301 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3361 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3421 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3481 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3541 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     3601 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     3661 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3721 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3781 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     3841 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3901 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4021 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4081 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4141 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4201 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4261 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4321 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4381 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4441 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4501 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4561 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4621 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4681 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4741 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4801 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4861 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4921 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4981 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5041 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5101 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5161 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5221 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5281 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5341 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5401 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5461 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5521 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5581 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5641 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5701 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5761 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5821 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5881 catttatcag ggtta
//
