LOCUS       sequence-364614-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven yellow fluorescent protein YPet 
            localized to the mitochondria (PhCMV-4xCOX8-YPet-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6342)
  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 6342)
  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: pTS2396-Tier1-PhCMV-4xCOX8_Ypet
FEATURES             Location/Qualifiers
     source          1..6342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        46..425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        426..629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     580..600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     674..693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        674..692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             745..816
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             838..912
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             934..1008
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1030..1104
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(1474..1493)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1939..1956)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1945..2169
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2215..2643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2302..2321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2512..2533
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2652..2672)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2657..2986
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2837..2972
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2899..2918
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3053..3847
                     /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(3107..3126)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3717..3736
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4021..4142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4058..4077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4112..4131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4191..4207)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4191..4207)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4204..4226)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4215..4231
                     /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(4239..4245,4246..4263,4264..4269))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4284..4305
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4422..4439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4593..5181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4673..4692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5352..6143,6144..6212))
                     /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     5975..5994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6213..6317)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgacg gatcgggaga tctccacgcg tggtaccctc gagttgacat tgattattga
       61 gtagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      121 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      181 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      241 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      301 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      361 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      421 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      481 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      541 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      601 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      661 ggcttatcga aattaatacg actcactata gggagaccca agctgttcga agcggaattc
      721 accatgacta gtggtggttc tggtagcgtg ctgacaccat tgttgctcag aggcctcact
      781 ggttcagcca ggcggctgcc agtcccaaga gctaagatcc atagtctggg agatccaatg
      841 tcagttctca ctcccctttt gcttagggga ctgaccggaa gcgctcggcg gttgcctgtc
      901 ccacgcgcca agattcactc cctgggcgac cctatgagtg tccttactcc tcttttgctg
      961 agaggcttga cagggagcgc tcggaggctc ccagtgcctc gcgcaaagat ccattctctc
     1021 ggagatccca tgtccgtcct cactcctctg ctgctgcgcg ggcttacagg ttccgctcgc
     1081 cgcctgcctg ttcctcgggc aaagatacac agtctgggcg atcctcaccg gcgctatctc
     1141 atgggcggtg cagctagtgg tggttctggt gtgagcaaag gcgaagagct gttcaccggc
     1201 gtggtgccca tcctggtgga gctggacggc gacgtgaacg gccacaagtt cagcgtgagc
     1261 ggcgagggcg agggcgacgc cacctacggc aagctgaccc tgaagctgct gtgcaccacc
     1321 ggcaagctgc ccgtgccctg gcccaccctg gtgaccaccc tgggctacgg cgtgcagtgc
     1381 ttcgcccggt accccgacca catgaagcag cacgacttct tcaagagcgc catgcccgag
     1441 ggctacgtgc aggagcggac catcttcttc aaggacgacg gcaactacaa gacccgggcc
     1501 gaggtgaagt tcgagggcga caccctggtg aaccggatcg agctgaaggg catcgacttc
     1561 aaggaggacg gcaacatcct gggccacaag ctggagtaca actacaacag ccacaacgtg
     1621 tacatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccggcacaac
     1681 atcgaggacg gcggcgtgca gctggccgac cactaccagc agaacacccc catcggcgac
     1741 ggccccgtgc tgctgcccga caaccactac ctgagctacc agagcgccct gttcaaggac
     1801 cccaacgaga agcgggacca catggtgctg ctggagttcc tgaccgccgc cggcatcacc
     1861 gagggcatgg acgagctgta caaggctagc ggatccaccg gtgtctagaa agctttgagg
     1921 ccggcctgca gggatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1981 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2041 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2101 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2161 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2221 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2281 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2341 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2401 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2461 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2521 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2581 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2641 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     2701 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2761 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2821 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2881 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     2941 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3001 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3061 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3121 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3181 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3241 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3301 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3361 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3421 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3481 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3541 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3601 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3661 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3721 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3781 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3841 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3901 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3961 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4021 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4081 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4141 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4201 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4261 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4321 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4381 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4441 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4501 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4561 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4621 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4681 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4741 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4801 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4861 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4921 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4981 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5041 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5101 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5161 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5221 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5281 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5341 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5401 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5461 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5521 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5581 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5641 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5701 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5761 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5821 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5881 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5941 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6001 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6061 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6121 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6181 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6241 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6301 caaatagggg ttccgcgcac atttccccga aaagtgccac ct
//
