LOCUS       sequence-79157-e        5651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains an N-terminal tandem affinity purification tag (relevant to
            any system) within a construct designed for genome integration into 
            trypanosomes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5651)
  AUTHORS   Schimanski B, Nguyen TN, Gunzl A
  TITLE     Highly efficient tandem affinity purification of trypanosome protein
            complexes based on a novel epitope combination.
  JOURNAL   Eukaryot Cell. 2005 Nov;4(11):1942-50.
  PUBMED    16278461
REFERENCE   2  (bases 1 to 5651)
  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: "Eukaryot 
            Cell."; date: "2005-11"; volume: "4(11)"; pages: "1942-50"
COMMENT     Sequence Label: pN-PURO-PTP
FEATURES             Location/Qualifiers
     source          1..5651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2702..3301
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2702..2721)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3198..3218
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4527..4700
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             4701..4874
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             4911..4931
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4938..4973
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     primer_bind     complement(5007..5026)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5008..5026)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5033..5050)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5033..5049)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5042..5064)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5191..5646
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5278..5297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5488..5509
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctcctaaag tcgaggaggt
     2221 tgactaagtt cccaggtgta tttcggaccg gtgctgcagt cgtctcactg tctacggtga
     2281 tggtccgccg ccacattttc tcttcgtctc tgtagcattt aggaaccctc gttacgggaa
     2341 agatgcgtga cacattgatg ctactactat tattaatact ctactattat tattattatt
     2401 attattttta ttttagttcc ccaccaccgt cattattatt atcattatta ttatcaatat
     2461 taccattatt attattacga taatgagcgt tagtgctatt accgctgttg ttgccatctt
     2521 tgttaccgtg gtgctttttt tcctttggtt catgcaactg cagcccggca tcgtcctccc
     2581 atacttgcac tcctgctctt ccttcctatt tgcatttgcc cacgtgcccc acgcgtgcat
     2641 cacataagca tcaaggaaca agcaacagct ataaaacaag gaatatctgc ctctttgaca
     2701 tatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt
     2761 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga
     2821 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     2881 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     2941 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3001 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3061 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     3121 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3181 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3241 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3301 atcgaacttc gaaaattcac ggaggcagag tccaacatga acgatctcgt gtctgagtac
     3361 cagcagtacc aggatgccac gattgaggag gagggcgagt tcgacgagga ggagcaatac
     3421 tagacgcgga cggggcattt cccgttcgtc attagcagta ggtaatgaag atgtttgttt
     3481 ctcgtcccct ttctccttcg tccttctgtc attttgttct tttgtgttta tgttttgttg
     3541 ttgttttctt taattttttt tttcttccac gtttgtgtac atccgcgcgc cactctattc
     3601 agagagccac ggatagtaga ggaggtggga agggtatatg agggacacgc gtaccatgat
     3661 gtgggatgta ttggggtccc tgtctgtcct tacgtgacta tgtatgaacc gtcacgtgta
     3721 agatgagcta gtgagatcaa cagtacaact catcaacacg ccttcttctc gttagatgta
     3781 cacaatcttg atcctccacc tttatgggtc ccattgtttg cctcttccgc tgtgtggagt
     3841 gcgcctacac gcacttctca cttcgtaagt ggtggtggcg taagtattgc ctaatgttga
     3901 ctctatgttc tcctctcctc accccctcgc ggtgctgatt tctgacagat cttcaaacac
     3961 tagtttaagc aaaggactat tcatccgttt atattagcaa cagtaggtac tagcaccact
     4021 aacaacaaca acaaagcact tctatttatt tatcaagctt ctgacctttg taccatcgct
     4081 gccctcacct gttcaactat ggtatttagt ggcctcagga ggttgcctgg gtggctgggt
     4141 ctttacttcg tcaggataat ttgaagttcg ttttccgtaa acagttaccc agttgtgagg
     4201 ttgttgcgac gcacattcac atgtcttcat cttatttgtt ggtatatttc cttgctgctc
     4261 gttttggggg tgcgggcgcg ctgattcaaa gagaggggag aatctgccgc actgcttgca
     4321 gcacaggtct tgctgtttcc cccctctatt gtcgtttttc acattcatgc tatttccggt
     4381 tgaagcggtc gttgtggccc tgccgggtgg ttctgcacat gctatgcact ttctccctct
     4441 tcttcatcct cgtaatgcta acagaataag ttgtggaaag cgtgaagcgt gcaaacatgg
     4501 caggccttgc gcaccacgat gaagccgtgg acaacaaatt caacaaagaa caacaaaacg
     4561 cgttctatga gatcttacat ttacctaact taaacgaaga acaacgaaac gccttcatcc
     4621 aaagtttaaa agatgaccca agccaaagcg ctaacctttt agcagaagct aaaaagctaa
     4681 atgatgctca ggcgccgaaa gtagacaaca aattcaacaa agaacaacaa aacgcgttct
     4741 atgagatctt acatttacct aacttaaacg aagaacaacg aaacgccttc atccaaagtt
     4801 taaaagatga cccaagccaa agcgctaacc ttttagcaga agctaaaaag ctaaatgatg
     4861 ctcaggcgcc gaaagtagac gcgaattgtg atatacctac aactgcttct gaaaatttat
     4921 attttcaagg tgaactagaa gatcaggtgg atcctcgtct tattgatggg aaagcggccg
     4981 ctacgtaggg cccggtaccc aattcgccct atagtgagtc gtattacaat tcactggccg
     5041 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5101 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5161 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5221 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5281 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5341 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5401 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5461 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5521 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5581 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5641 caatttaggt g
//
