LOCUS       sequence-364218-        5610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven four-fold repeat of a 
            mitochondrial matrix localization signal derived from mitochondrial 
            matrix protein Cox8 (PhCMV-4xCox8-pA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5610)
  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 5610)
  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: pTS1051-Tier1-PhCMV-4xCox8
FEATURES             Location/Qualifiers
     source          1..5610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     743..762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        743..761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             814..885
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             907..981
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1003..1077
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1099..1173
                     /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(1276..1293)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1282..1506
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1552..1980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1639..1658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1849..1870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1989..2009)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1994..2323
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2174..2309
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2236..2255
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2390..3184
                     /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(2444..2463)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3054..3073
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3358..3479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3395..3414)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3449..3468
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3528..3544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3528..3544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3541..3563)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3552..3568
                     /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(3576..3582,3583..3600,3601..3606))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3621..3642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3759..3776)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3930..4518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4010..4029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4689..5480,5481..5549))
                     /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     5312..5331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5550..5610,1..44))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
       61 gtgccacctg acgtcgacgg atcgggagat ctccacgcgt ggtaccctcg agttgacatt
      121 gattattgag tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      721 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctgttcgaa
      781 gcggaattca ccatgactag tggtggttct ggtagcgtgc tgacaccatt gttgctcaga
      841 ggcctcactg gttcagccag gcggctgcca gtcccaagag ctaagatcca tagtctggga
      901 gatccaatgt cagttctcac tccccttttg cttaggggac tgaccggaag cgctcggcgg
      961 ttgcctgtcc cacgcgccaa gattcactcc ctgggcgacc ctatgagtgt ccttactcct
     1021 cttttgctga gaggcttgac agggagcgct cggaggctcc cagtgcctcg cgcaaagatc
     1081 cattctctcg gagatcccat gtccgtcctc actcctctgc tgctgcgcgg gcttacaggt
     1141 tccgctcgcc gcctgcctgt tcctcgggca aagatacaca gtctgggcga tcctcaccgg
     1201 cgctatctca tgggcggtgc agctagcgga tccaccggtg tctagaaagc tttgaggccg
     1261 gcctgcaggg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1321 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1381 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     1441 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     1501 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     1561 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1621 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1681 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1741 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1801 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1861 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1921 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1981 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2041 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2101 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2161 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2221 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     2281 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     2341 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     2401 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     2461 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     2521 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     2581 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     2641 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     2701 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     2761 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     2821 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     2881 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     2941 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3001 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3061 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3121 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     3181 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     3241 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3301 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3361 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3421 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3481 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     3541 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3601 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3661 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3721 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3781 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3841 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3901 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3961 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4021 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4081 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4141 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4201 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4261 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4321 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4381 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4441 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4501 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4561 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4621 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4681 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4741 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4801 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4861 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4921 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4981 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5041 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5101 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5161 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5221 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5281 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5341 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5401 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5461 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5521 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5581 tatcagggtt attgtctcat gagcggatac
//
