LOCUS       40924_42924        7515 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTcTAPN, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, 
            Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     A high-throughput cloning system for reverse genetics in Trypanosoma
            cruzi
  JOURNAL   BMC Microbiol. 10 (1), 259 (2010)
  PUBMED    20942965
REFERENCE   2  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PAF., Fragoso SP, Probst CM, Preti 
            H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-APR-2010) Instituto Carlos Chagas, Professor Algacyr 
            Munhoz Mader, 3775, Curitiba, Parana 81350010, Brasil
REFERENCE   3  (bases 1 to 7515)
  AUTHORS   Batista M, Marchini FK, Celedon PAF., Fragoso SP, Probst CM, Preti 
            H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-NOV-2010) Instituto Carlos Chagas, Professor Algacyr 
            Munhoz Mader, 3775, Curitiba, Parana 81350010, Brasil
REFERENCE   4  (bases 1 to 7515)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7515)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Microbiol."; date: "2010"; volume: "10"; issue: "1"; pages: "259"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (28-APR-2010) Instituto Carlos Chagas, Professor Algacyr Munhoz 
            Mader, 3775, Curitiba, Parana 81350010, Brasil"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (18-NOV-2010) Instituto Carlos Chagas, Professor Algacyr Munhoz 
            Mader, 3775, Curitiba, Parana 81350010, Brasil"
COMMENT     SGRef: number: 4; type: "Journal Article"
COMMENT     On Nov 18, 2010 this sequence version replaced HM162842.1.
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      659..1275
                     /note="derived from Trypanosoma cruzi 18S ribosomal RNA
                     gene promoter region"
                     /regulatory_class="promoter"
     CDS             1977..1997
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             2043..2120
                     /label=CBP
                     /note="calmodulin-binding peptide"
     protein_bind    complement(2136..2169)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2223..2342
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2372..2402
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2456..3112
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     CDS             3457..3759
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     protein_bind    complement(3803..3927)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4227..5015
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
     promoter        complement(5327..5345)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5366..5382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5390..5406)
                     /label=lac operator
                     /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(5414..5444)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5459..5480)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5768..6356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6530..7387)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7388..7492)
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccagtttcct tgcggggctt atttggtgtg ttggattttt aattttttga tgattcggag
      721 gaattttttg caatttttgg gtacccctaa aaaaattttt ggagtttccg gggttcgatc
      781 caacttgtgc ttttttaacc gtccatttgt gtctagtaca tcatggagcg gtattctctc
      841 cggtggccca ggtggcgtcg gaaggaattg gtgtccctca ggatgtttgg tttttcgttt
      901 ttttttttgc ggtcttttgt ttcgcatcgc cgtatcgtgt gcggcgcgac cttctttttg
      961 gctcattggt cagtgaggtt tataataaag tggatcattt gggtttttgg ccctttgcgg
     1021 cgcacccgcg gaatggggga gggattttca tataatgtag tgtacattgt gtgtgggtcg
     1081 caaaatgtca tgccgtgtgc aatgtttatt tgcgtgctgt gcgtgtcctt ttgtgattcc
     1141 accacttgtg tgtattttac cttatcgtct ctccctgccg ttggctgtgc gtgaattgtg
     1201 cttcgcattc gtgggaaatc aaaaggggaa tcaacgtgtt cttttttttc agaaggggag
     1261 tcaaagcgcc gtgtggatat ctctgcgatg ctgtggaccg acgcattgaa atacacaccg
     1321 tctttggcgt tccttttttt ttttttgatt tgtttttttt attgagatgt tttgttttgt
     1381 ttttttcagt ttttattata tcagcagttt gtccgcctgc attcatgcag tgtttggtaa
     1441 ttctttctat tttttggaat tatggcgata atattcttgt ctttaaactt cttataacca
     1501 attgtgcttt agagtttcct gcttagttgc tattaacaca ctgttaggaa cgtgaaacca
     1561 tggcatgcat gaaagctgat gcgcaaaata acttcaacaa agatcaacaa agcgccttct
     1621 atgaaatctt gaacatgcct aacttaaacg aagcgcaacg taacggcttc attcaaagtc
     1681 ttaaagacga cccaagccaa agcactaacg ttttaggtga agctaaaaaa ttaaacgaat
     1741 ctcaagcacc gaaagctgat aacaatttca acaaagaaca acaaaatgct ttctatgaaa
     1801 tcttgaatat gcctaactta aacgaagaac aacgcaatgg tttcatccaa agcttaaaag
     1861 atgacccaag ccaaagtgct aacctattgt cagaagctaa aaagttaaat gaatctcaag
     1921 caccgaaagc ggataacaaa ttcaacaaag aatctagtac cccaactact gcttctgaaa
     1981 atctatattt tcaaggtgaa ctaaaaactg ctgctttggc tcaacatgcg ctcgagaaga
     2041 tgaagcgacg atggaaaaag aatttcatag ccgtctcagc agccaaccgc tttaagaaaa
     2101 tctcatcctc cggggcactt ggaggagggg tcgacataac ttcgtatagc atacattata
     2161 cgaagttatc tgcagccaat agcaccaagg acgtccctat ctacatattt atatctagaa
     2221 tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat
     2281 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     2341 catattggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
     2401 tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga
     2461 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     2521 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     2581 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     2641 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     2701 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     2761 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     2821 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     2881 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     2941 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     3001 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     3061 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg
     3121 tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg
     3181 tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag
     3241 cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg
     3301 tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg
     3361 aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga
     3421 acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat
     3481 aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc
     3541 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
     3601 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
     3661 gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     3721 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata
     3781 cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac agcattatgt
     3841 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
     3901 tcgttcagct ttcttgtaca aagtggtgat actagttagt ctgcgatgct gtggaccgac
     3961 gcattgaaat acacaccgtc tttggcgttc cttttttttt ttttgatttg ttttttttat
     4021 tgagatgttt tgttttgttt ttttcagttt ttattatatc agcagtttgt ccgcctgcat
     4081 tcatgcagtg tttggtaatt ctttctattt tttggaatta tggcgataat attcttgtct
     4141 ttaaacttct tataaccaat tgtgctttag agtttcctgc ttagttgcta ttaacacact
     4201 gttaggaacg tgaaaccctc gagatgattg aacaagatgg attgcacgca ggttctccgg
     4261 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4321 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4381 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4441 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4501 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4561 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4621 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4681 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4741 ggctcaaggc gcgaatgccc gacggcgatg atctcgtcgt gacccatggc gatgcctgct
     4801 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4861 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4921 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4981 gcatcgcctt ctatcgcctt cttgacgagt tcttctgact cgagtctgcg atgctgtgga
     5041 ccgacgcatt gaaatacaca ccgtctttgg cgttcctttt tttttttttg atttgttttt
     5101 tttattgaga tgttttgttt tgtttttttc agtttttatt atatcagcag tttgtccgcc
     5161 tgcattcatg cagtgtttgg taattctttc tattttttgg aattatggcg ataatattct
     5221 tgtctttaaa cttcttataa ccaattgtgc tttagagttt cctgcttagt tgctattaac
     5281 acactgttag gaacgtgaaa ccgggcccgg tacccagctt ttgttccctt tagtgagggt
     5341 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5401 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5461 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5521 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5581 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5641 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5701 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5761 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5821 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5881 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5941 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6001 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6061 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6121 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6181 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6241 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6301 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6361 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6421 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6481 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6541 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6601 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6661 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6721 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6781 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6841 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6901 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6961 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7021 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7081 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7141 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7201 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7261 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7321 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7381 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7441 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7501 cccgaaaagt gccac
//
