LOCUS       sequence-364611-        5979 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 
            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: pTS2392-Tier1-PhCMV-p2a_medFT
FEATURES             Location/Qualifiers
     source          1..5979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..32
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(151..171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        156..485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      336..471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     398..417
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             552..1346
                     /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(606..625)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1216..1235
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1520..1641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1557..1576)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1611..1630
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1690..1706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1690..1706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1703..1725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1714..1730
                     /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(1738..1744,1745..1762,1763..1768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1783..1804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1921..1938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2092..2680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2172..2191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2851..3642,3643..3711))
                     /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     3474..3493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3712..3816)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3887..4266
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4267..4470
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4421..4441
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4515..4534
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4515..4533
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4595..4651
                     /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             4658..5362
                     /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(4800..4818)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5068..5087
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5417..5434)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5423..5647
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5693..5979,1..142)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5780..5799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
       61 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      121 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
      181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
      241 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
      301 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
      361 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
      421 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
      481 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
      541 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
      601 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
      661 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
      721 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
      781 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
      841 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
      901 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
      961 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1021 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1081 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     1141 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     1201 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     1261 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     1321 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     1381 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     1441 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     1501 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     1561 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1621 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     1681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2521 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3841 tgacgtcgac ggatcgggag atctccacgc gtggtaccct cgagttgaca ttgattattg
     3901 agtagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3961 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     4021 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     4081 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     4141 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     4201 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     4261 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     4321 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     4381 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     4441 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
     4501 tggcttatcg aaattaatac gactcactat agggagaccc aagctgttcg aagcggaatt
     4561 caccatgact agtggtggtt ctggtggaag cggagctact aacttcagcc tgctgaagca
     4621 ggctggtgac gtggaggaga accctggacc tgctagtgtg agcaagggcg aggaggataa
     4681 catggccatc atcaaggagt tcatgcgttt caaggtgcac ctggagggct ccgtggacgg
     4741 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagagcgc
     4801 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     4861 gttcatgtac ggctccaggg cctacgtgaa gcaccccgcc gacatccccg actactggaa
     4921 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg atggcggcgt
     4981 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     5041 gcgcggcacc aacttccctt ccgacggccc cgtaatgcag aagaagacca tgggctggga
     5101 ggcctcctcc gagcggatat accccgagga cggcgccctg aagggcgaga tcaagcagag
     5161 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     5221 gaagcccgtg ctgctgcccg gcgcctacaa cgtcaacatc aagatggaca tcacctccca
     5281 caacgaggac tacaccatcg ttgaacagtg cgaacgcgcc gagggccacc attccaccgg
     5341 cggcatggac gagctgtaca aggctagcgg atccaccggt gtctagaaag ctttgaggcc
     5401 ggcctgcagg gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5461 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5521 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5581 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     5641 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     5701 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5761 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5821 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5881 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5941 tgatagacgg tttttcgccc tttgacgttg gagtccacg
//
