LOCUS       sequence-364518-        5895 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 
            (PhCMV-fastFT-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: pTS2385-Tier1-PhCMV-fastFT
FEATURES             Location/Qualifiers
     source          1..5895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        47..426
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        427..630
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     581..601
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     675..694
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        675..693
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             734..1438
                     /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(876..894)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1144..1163
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1493..1510)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1499..1723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1769..2197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1856..1875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2066..2087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2206..2226)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2211..2540
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2391..2526
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2453..2472
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2607..3401
                     /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(2661..2680)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3271..3290
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3575..3696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3612..3631)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3666..3685
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3745..3761)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3745..3761)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3758..3780)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3769..3785
                     /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(3793..3799,3800..3817,3818..3823))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3838..3859
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3976..3993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4147..4735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4227..4246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4906..5697,5698..5766))
                     /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     5529..5548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5767..5871)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacgtcgac ggatcgggag atctccacgc gtggtaccct cgagttgaca ttgattattg
       61 agtagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      121 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      181 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      241 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      301 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      361 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      421 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      481 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      541 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      601 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
      661 tggcttatcg aaattaatac gactcactat agggagaccc aagctgttcg aagcggaatt
      721 caccatgact agtgtgagca agggcgagga ggataacatg gccatcatca aagagttcat
      781 gcgtttcaaa gtgcacgtgg agggttccgt gaacggccac gagttcgaga tcgagggcga
      841 gggcaagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      901 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccagggccta
      961 cgtgaagcat cccgccgaca tccccgacta ctggaagctg tccttccccg agggcttcaa
     1021 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     1081 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggaactaact tcccctccga
     1141 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc ggatgtaccc
     1201 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     1261 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     1321 ttacaacgtc aatatcaagt tggacatcac atcccacaac gaggactaca ccatcgtgga
     1381 acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaaggc
     1441 tagcggatcc accggtgtct agaaagcttt gaggccggcc tgcagggatc agcctcgact
     1501 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1561 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1621 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1681 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     1741 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1801 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1861 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1921 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1981 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2041 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2101 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2161 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     2221 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2281 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2341 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2401 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2461 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2521 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     2581 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2641 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2701 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2761 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     2821 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2881 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2941 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3001 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3061 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3121 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3181 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3241 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3301 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3361 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3421 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3481 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3541 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3601 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3661 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3721 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3781 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3841 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3901 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3961 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4021 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4081 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4141 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4201 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4261 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4321 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4381 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4441 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4501 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4561 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4621 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4681 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4741 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4801 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4861 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4921 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4981 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5041 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5101 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5161 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5221 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5281 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5341 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5401 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5461 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5521 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5581 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5641 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5701 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5761 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5821 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5881 ccgaaaagtg ccacc
//
