LOCUS       sequence-102758-        5451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCR2.1 linker-tev-linker-3X-FLAG::KanMX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5451)
  AUTHORS   Williams CC, Jan CH, Weissman JS
  TITLE     Targeting and plasticity of mitochondrial proteins revealed by 
            proximity-specific ribosome profiling.
  JOURNAL   Science. 2014 Nov 7;346(6210):748-51. doi: 10.1126/science.1257522.
  PUBMED    25378625
REFERENCE   2  (bases 1 to 5451)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2014-11-7- 7"; volume: "346"; issue: 
            "6210"; pages: "748-51"
COMMENT     Sequence Label: pJW1514
FEATURES             Location/Qualifiers
     source          1..5451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     gene            complement(393..1749)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(393..590)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(596..1405)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1319..1338
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1406..1749)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      complement(1874..2121)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2087..2105
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     2123..2139
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2124..2140
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2146..2217)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             complement(2230..2250)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2338..2357)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2339..2357)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2364..2381)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2364..2380)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2373..2395)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2518..2820
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2699..2718
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3169..3963
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3169..3963
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3223..3242)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3833..3852
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4164..4173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4170..4544
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      4682..5270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5171..5190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5424..5441
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttatcg atgaattcga gctcgtttaa
      361 actggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat tcacatacga
      421 ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg
      481 cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac aatataaaaa
      541 aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt actgattaga
      601 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
      661 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
      721 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
      781 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
      841 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat
      901 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
      961 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1021 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1081 ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat gcatcatcag
     1141 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1201 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1261 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1321 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     1381 aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt gagaactgta
     1441 tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca aatcctaacc
     1501 ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg tgaggggagc
     1561 gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg ccgtgcggcc
     1621 atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa tgtacgggcg
     1681 acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg tattctgggc
     1741 ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaacag atctggcgcg
     1801 ccttaattaa cccggggatc cgtcgacctg cagcgtacga agcttcagct ggcggccgcg
     1861 ttcggtaccg gccgcaaatt aaagccttcg agcgtcccaa aaccttctca agcaaggttt
     1921 tcagtataat gttacatgcg tacacgcgtc tgtacagaaa aaaaagaaaa atttgaaata
     1981 taaataacgt tcttaatact aacataacta taaaaaaata aatagggacc tagacttcag
     2041 gttgtctaac tccttccttt tcggttagag cggatgtggg gggagggcgt gaatgtaagc
     2101 gtgacataac taattacatg actcgaggtc gacggtatcg atttacttgt catcgtcatc
     2161 tttataatcc ttgtcatcgt catctttata atccttgtca tcgtcatctt tataatcaga
     2221 acctgaacca gattgaaaat acaaattttc acctgaacct gaacctccaa gggcgaattc
     2281 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc caattcgccc
     2341 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2401 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2461 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata cgtacggcag
     2521 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     2581 gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     2641 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     2701 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     2761 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     2821 atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac gtagaaagcc
     2881 agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg
     2941 gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta
     3001 gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt
     3061 aaggttggga agccctgcaa agtaaactgg atggctttct cgccgccaag gatctgatgg
     3121 cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3181 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3241 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3301 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     3361 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3421 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3481 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3541 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3601 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3661 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     3721 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3781 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3841 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3901 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3961 tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     4021 atttcacacc gcatacaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4081 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4141 ttcaataata gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc
     4201 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     4261 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     4321 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     4381 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     4441 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     4501 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc
     4561 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4621 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4681 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4741 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4801 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4861 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4921 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4981 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5041 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5101 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5161 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5221 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5281 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg
     5341 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5401 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
