LOCUS       sequence-194172-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CLIP-Tagged Cox8A in Mammalian cells .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  AUTHORS   Ana Egana
  TITLE     Egana Lab NEB plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6078)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCLIPf-Cox8A Control Plasmid
FEATURES             Location/Qualifiers
     source          1..6078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(157..174)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     384..403
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             599..1402
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(662..681)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1272..1291
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1532..1653
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1569..1588)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1755..1771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1755..1771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1768..1790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1779..1795
                     /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(1803..1809,1810..1827,1828..1833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1848..1869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1986..2003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2157..2745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2237..2256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2916..3707,3708..3776))
                     /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     3539..3558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3777..3881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3956..3975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4147..4526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4527..4730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4681..4701
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4775..4794
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4775..4793
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4842..4916
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             5097..5642
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     intron          5766..5995
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     5978..5997
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(6074..6078,1..563)
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
ORIGIN
        1 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
       61 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
      121 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
      181 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
      241 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
      301 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
      361 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
      421 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
      481 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
      541 tcctttgaaa aacacgatga taagcttgcc acaacccggg ataattcctg cagccaatat
      601 gggatcggcc attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
      661 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
      721 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
      781 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
      841 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
      901 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
      961 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1021 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     1081 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     1141 gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     1201 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     1261 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     1321 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     1381 ccttcttgac gagttcttct gaggggatca attctctaga taactgatca taatcagcca
     1441 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1501 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1561 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1621 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgtc gagtgcattc tagttgtggt
     1681 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     1741 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1801 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1861 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3901 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     3961 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     4021 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     4081 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     4141 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     4201 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     4261 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     4321 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     4381 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     4441 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     4501 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     4561 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     4621 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     4681 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     4741 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     4801 aagcttggta ccgagctcgg atcgatggcc gcaagcttac catgtccgtc ctgacgccgc
     4861 tgctgctgcg gggcttgaca ggctcggccc ggcggctccc agtgccgcgc gccaagatcc
     4921 attcgttgcc gccggagggg aagcttggga tcatggaatt ggccgttggg cttacctcct
     4981 gcttcgtgac cttcctcctg ccagcgggct ggatcctgtc acacctggag acctacagga
     5041 ggccagagga tatcggcgcg ccagcattta aatctgtaca gaccggtgaa ttcaccatgg
     5101 acaaagactg cgaaatgaag cgcaccaccc tggatagccc tctgggcaag ctggaactgt
     5161 ctgggtgcga acagggcctg caccgtatca tcttcctggg caaaggaaca tctgccgccg
     5221 acgccgtgga agtgcctgcc ccagccgccg tgctgggcgg accagagcca ctgatccagg
     5281 ccaccgcctg gctcaacgcc tactttcacc agcctgaggc catcgaggag ttccctgtgc
     5341 cagccctgca ccacccagtg ttccagcagg agagctttac ccgccaggtg ctgtggaaac
     5401 tgctgaaagt ggtgaagttc ggagaggtca tcagcgagag ccacctggcc gccctggtgg
     5461 gcaatcccgc cgccaccgcc gccgtgaaca ccgccctgga cggaaatccc gtgcccattc
     5521 tgatcccctg ccaccgggtg gtgcagggcg acagcgacgt ggggccctac ctgggcgggc
     5581 tcgccgtgaa agagtggctg ctggcccacg agggccacag actgggcaag cctgggctgg
     5641 gtcctgcagg cggatccgcg tttaaactcg aggttaatta atgagcggcc gcttaagccg
     5701 gccgcataga taactgatcc agtgtgctgg aattaattcg ctgtctgcga gggccagctg
     5761 ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat tgtcagtttc
     5821 caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga gggtggccgc
     5881 gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg gcaggcttga
     5941 gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc cacaggtgtc
     6001 cactcccagg tccaactgca ggtcgagcat gcatctaggg cggccaattc cgcccctctc
     6061 cccccccccc ttttccct
//
