LOCUS       sequence-79156-e        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  contains a C-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 6662)
  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 6662)
  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: pC-PTP-NEO
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1421..1456
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             1481..1501
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1541..1714
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             1715..1888
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     CDS             2902..3696
                     /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(2956..2975)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3566..3585
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        complement(4474..4492)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4513..4529)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4513..4529)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4526..4548)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4537..4553
                     /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(4561..4567,4568..4585,4586..4591))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4606..4627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4744..4761)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4915..5503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4995..5014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5674..6465,6466..6534))
                     /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     6297..6316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6535..6639)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccctaacag agcaggccac ccccaccgtt tgtcccccag tgccgccgac gtcgtgagcc
      721 aatagcttcg agtccggtcc atatacttca caagcgggca ctggagcttg ttgccgctga
      781 gcgaccgcct gtggtttcgt cgtcgtcaat gaatgcgtgg cccgttgcag cagcagagaa
      841 atctgaccct aagcgttcgg cctggttgga gcagatgaag tcggagatga atcgccgcaa
      901 tccgtataca gatttgaata tatgctcgga cccgcggtgc acagttgtga tgtcggggtt
      961 gcacgccaaa acggtggagg aggatatccg catcttcagc gagcagtttg gtcgtgtggt
     1021 cagcactcgt ctaatttttg accgtaatgg tcacagccga cggtatggct ttgtgcagtt
     1081 tggacgggag gcggatgtgc agagagcagt tgcaagtagt ggaaagaggc ggcttcacgg
     1141 tcggagtgtt gtgatcgacg tggagcgagg gcggcaggaa cctgagtttc tgccgaaacg
     1201 cattgcaaaa gctgtgaaat tgcaggggac gtcgatcaat tcccccgttt tgccgtctgg
     1261 agacacgcca attggcaagc gccagcgcaa tgagaccgaa cggcaacaag atcgatcaag
     1321 ggaggctcca actaagccgg agccatcagg aggtgaactg agacaccgtg atgatgtgga
     1381 catccttctg gatgatatca tgagtattac gcggccgctg gaagatcagg tggatcctcg
     1441 tcttattgat gggaaatatg atattccaac tactgctagc gagaatttgt attttcaggg
     1501 tgagctcaaa accgcggctc ttgcgcaaca cgatgaagcc gtggacaaca aattcaacaa
     1561 agaacaacaa aacgcgttct atgagatctt acatttacct aacttaaacg aagaacaacg
     1621 aaacgccttc atccaaagtt taaaagatga cccaagccaa agcgctaacc ttttagcaga
     1681 agctaaaaag ctaaatgatg ctcaggcgcc gaaagtagac aacaaattca acaaagaaca
     1741 acaaaacgcg ttctatgaga tcttacattt acctaactta aacgaagaac aacgaaacgc
     1801 cttcatccaa agtttaaaag atgacccaag ccaaagcgct aaccttttag cagaagctaa
     1861 aaagctaaat ggtgctcagg cgccgaaagt agacgcgaat tccgcgggga agtcaacctg
     1921 atcgcctgaa ccgctatttt aagtatatat atatgtatat tacctttttt tgttttttct
     1981 ttgcgctttt acctcctctt ttgtctcaac tttttttaat tttatgagag gcacttgtgt
     2041 gaaattggac accataaagg gctgaggagg gaagcagaga gcaaatgaaa atatgggagg
     2101 gtgaaaaata catacatata tatttgtgtt tgttcgtatt tggagggata caaatacgat
     2161 atatgaagat atcaacagtt tttcacctgt gggggaagcg tgattattat cagtgagtgg
     2221 atggactgaa gaacgaataa atgtgtaaca atttcgccct tcattttttc ttcggtggca
     2281 gacgggttta aatctaatcg gataaatagc actaaagtaa cgaatgttaa aataaccacc
     2341 gcaaaggcat accttttcct tttttgccca ttgtggtggg aagtcagtgg catcgataag
     2401 cttcctaaag tcgaggaggt tgactaagtt cccaggtgta tttcggaccg gtgctgcagt
     2461 cgtctcactg tctacggtga tggtccgccg ccacattttc tcttcgtctc tgtagcattt
     2521 aggaaccctc gttgcgggaa agatgcgtga cacattgatg ctactactat tattaatact
     2581 ctactattat tattattatt attattttta ttttagttcc ccaccaccgt cattattatt
     2641 atcattatta ttatcaatat taccattatt attattacga taatgagcgt tagtgctatt
     2701 accgctgttg ttgccatctt tgttaccgtg gtgctttttt tcctttggtt catgcaactg
     2761 cagcccggca tcgtcctccc atacttgcac tcctgctctt ccttcctatt tgcatttgcc
     2821 cacgtgcccc acgcgtgcat cacataagca tcaaggaaca agcaacagct ataaaacaag
     2881 gaatatctgc ctctttgaca tatgattgaa caagatggat tgcacgcagg ttctccggcc
     2941 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3001 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3061 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3121 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3181 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3241 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3301 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3361 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3421 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3481 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3541 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3601 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3661 atcgccttct atcgccttct tgacgagttc ttctgaggat ccgttaactt cgaaaattca
     3721 cggaggcaga gtccaacatg aacgatctcg tgtctgagta ccagcagtac caggatgcca
     3781 cgattgagga ggagggcgag ttcgacgagg aggagcaata ctagacgcgg acggggcatt
     3841 tcccgttcgt cattagcagt aggtaatgaa gatgtttgtt tctcgtcccc tttctccttc
     3901 gtccttctgt cattttgttc ttttgtgttt atgttttgtt gttgttttct ttaatttttt
     3961 ttttcttcca cgtttgtgta catccgcgcg ccactctatt cagagagcca cggatagtag
     4021 aggaggtggg aagggtatat gagggacacg cgtaccatga tgtgggatgt attggggtcc
     4081 ctgtctgtcc ttacgtgact atgtatgaac cgtcacgtgt aagatgagct agtgagatca
     4141 acagtacaac tcatcaacac gccttcttct cgttaaatgt acacaatctt gatcctccac
     4201 ctttatgggt cccattgttt gcctcttccg ctgtgtggag tgcgcctaca cgcacttctc
     4261 acttcgtaag tggtggtggc gtaagtattg cctaatgttg actctatatt ctcctctcct
     4321 caccccctcg cggtgctgat ttctgacaga tcttcaaaca ctagtttaag caaaggacta
     4381 ttcatccgtt tatattagca acagtaggta ctagcaccac taacaacaac aacaaagcac
     4441 ttctatttat ttatcgagct ccagcttttg ttccctttag tgagggttaa tttcgagctt
     4501 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4561 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4621 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4681 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4741 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4801 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4861 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4921 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4981 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5041 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5101 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5161 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5221 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5281 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5341 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5401 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5461 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5521 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5581 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5641 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5701 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5761 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5821 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5881 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5941 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6001 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6061 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6121 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6181 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6241 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6301 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6361 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6421 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6481 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6541 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6601 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6661 ac
//
