LOCUS       sequence-125636-        7181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tdTomato in pTREX-b vector which confers resistance to 
            blasticidin. This vector is used for cloning a specific sgRNA by 
            BamHI, to transfect Cas9-expressing Trypanosoma cruzi 
            epimastigotes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7181)
  AUTHORS   Lander N, Li ZH, Niyogi S, Docampo R
  TITLE     CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 
            Genes in Trypanosoma cruzi Reveals Their Role in Flagellar 
            Attachment.
  JOURNAL   MBio. 2015 Jul 21;6(4). pii: e01012-15. doi: 10.1128/mBio.01012-15.
  PUBMED    26199333
REFERENCE   2  (bases 1 to 7181)
  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: "MBio."; 
            date: "2015-07-21"; volume: "6(4). pii: e01012-15. doi"; pages: " 
            10.1128/mBio.01012-15"
COMMENT     Sequence Label: tdTomato/pTREX-b
FEATURES             Location/Qualifiers
     source          1..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2477..2493)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2478..2494)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             3183..3581
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(4344..4362)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4383..4399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4383..4399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4396..4418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4407..4423
                     /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(4431..4437,4438..4455,4456..4461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4476..4497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4614..4631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4785..5373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4865..4884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5544..6335,6336..6404))
                     /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     6167..6186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6405..6509)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6535..6990)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6672..6693)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6884..6903
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7117..7139
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7131..7148
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7132..7148
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7155..7174
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7155..7173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagctcggtg caccctggga tatttgcgca cccacctttc ccgtcgcaat cggcgccgtc
       61 cgaacgcgga aatgttccga gaataatacg gctgggctgc gccgcgcgtt ccccgcgtga
      121 accaactatt gtgcgatgtg cagcatccta ttgtggtgct ttaccgcaca ccagtttcct
      181 tgcggggctt atttggtgtg ttggattttt aattttttga tgattcggag gaattttttg
      241 caatttttgg gtacccctaa aaaaattttg gagtttccgg ggttcgatcc aacttgtgct
      301 tttttaaccg tccatttgtg tctagtacat catggagcgg tattctctcc ggtggcccag
      361 gtggcgtcgg aaggaatcgg tgtccctcag gatgtttggt ttttcgtttt tttttttgcg
      421 gtcttttgtt tcgcatcgcc gtatcgtgtg cggcgcgacc ttctttttgg ctcattggtc
      481 agtgaggttt ataataaagt ggatcatttg ggtttttggc cctttgcggc gcacccgcgg
      541 aatgggggag ggattttcat ataattgtag tgtacacatt gtgtgtgggt cgcaaaatgt
      601 catgccgtgt gcaatgttta tttgcgtgct gtgcgtgtcc ttttgtgatt ccaccacttg
      661 tgtgtatttt accttatcgt ctctccctgc cgttggctgt gcgtgaattg tgcttcgcat
      721 tcgtgggaaa tcaaaagggg aatcaacgtg ttcttttttt ttcagaaggg gagtcaaagc
      781 gccgtgtgga tgccaagtct gtgcaagagt tctagtggat cccccaacga gtttcttcaa
      841 aatatgcagc ggattcacta agaaacattt tcacgcacga aagcgaaatt attatgattg
      901 ttattataat actttttctt tgttgtttta tccacttatt attgttgtgt taaattgttt
      961 ttaccttttt tcttttccaa cttcttttat gatgtctttt cttttttttt tttgctctat
     1021 aagttgtctt gtctagaatg gtttccaagg gtgaggaggt catcaaagag ttcatgcgct
     1081 tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
     1141 agggccgccc ctacgaaggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc
     1201 tacccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga
     1261 agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg
     1321 agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc
     1381 aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc
     1441 ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg
     1501 acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc
     1561 tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact
     1621 acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1681 acgagcgctc cgagggccgc caccacctgt tcctggggca tggcaccggc agcaccggca
     1741 gcggcagctc cggcaccgcc tcctccgagg acaacaacat ggccgtcatc aaagagttca
     1801 tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg
     1861 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     1921 gccccctgcc cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccaaggcgt
     1981 acgtgaagca ccccgccgac atccccgatt acaagaagct gtccttcccc gagggcttca
     2041 agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt gaccgtgacc caggactcct
     2101 ccctgcagga cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg
     2161 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc
     2221 cccgcgacgg cgtgctgaag ggcgagatcc accaggccct gaagctgaag gacggcggcc
     2281 actacctggt ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct
     2341 actactacgt ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     2401 aacagtacga gcgctccgag ggccgccacc acctgttcct gtacggcatg gacgagttgt
     2461 acaagtaaaa gcttatcgat accgtcgacc tcgagccatt tacgactcca aggcaacgct
     2521 gcagaacaac ctgccgaagg agcgccgctt cttcaagatt gtgtcgtggt acgacaacga
     2581 gtggggatac tcccaccgcg tggtggacct tgtacgccac atggcctcga aggatcgttc
     2641 ggcaaggttg taggcgtggc gatgacttca ggtctttctt ttgcgaatag ggatcttata
     2701 atacacgatg cgtgtcccgt gatgatcgtt accggtgctg ccacgatcca attgacacag
     2761 cgtcaagagc aaaacaattt tacttttccc tttaaggaca acaacaaaaa aatatataac
     2821 ttttttttct tttttttttt ttttgaaatt atatttatgg tcatctttgg gaaacaaaaa
     2881 gcagcaattt aatgatgcgg aaggatgagt gaaataatgt ttaatcaatg tacgaggatt
     2941 tggggtattg caaggaaaat gtagatgatt taattgggtg tgtgatgcag cttgtggtaa
     3001 tttttgctca cttccctttt tgccacatct ttttagtttt tctgcttttc tttccccatt
     3061 attccacttg tctctctttt cccacgtttc ctgcacgaat gcagaaagtg atatttttac
     3121 tttgaaagcc atctaccaac aacaattaca ttgaacagaa tttgggattg cgagatcaat
     3181 tcatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     3241 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     3301 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     3361 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     3421 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     3481 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     3541 ttcgtgaatt gctgccctct ggttatgtgt gggagggcta agaattgatc tccgccgaag
     3601 gagcgccgct tcttcaagat tgtgtcgtgg tacgacaacg agtggggata ctcccaccgc
     3661 gtggtggacc ttgtacgcca catggcctcg aaggatcgtt cggcaaggtt gtaggcgtgg
     3721 cgatgacttc aggtctttct tttgcgaata gggatcttat aatacacgat gcgtgtcccg
     3781 tgatgatcgt taccggtgct gccacgatcc aagtgacaca gtgtcaagag caaaacaatt
     3841 ttacttttcc ctttaaggac aacaacaaaa aaatatataa cttttttttc tttttttttt
     3901 ttgaaattat atttatggtc atctttggga aacaaaaagc agcaatttaa tgatgcggaa
     3961 ggatgagtga aataatgttt aatcaatgta cgaggatttg gggtattgca aggaaaatgt
     4021 agatgatttg attgggtgta tgatgcagct tgtggtaatt ttttacaact ttgcgttatt
     4081 ggaagatttt ggttgttcag tttctttttg tttataactg actgttttaa tttagtgtga
     4141 tttttttttt ttaaggattt aagctgtttt tctttttttt tctttttctt cactgatggt
     4201 gtttaaggtg tagtttagtg tttgtgcatt atgcgtcgta gtgttgttgt tctttggact
     4261 ttttcgaaca atactgggca ttatagccca cctgtgtata tgccgctaga gtatgctagc
     4321 cgtgtgggta ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca
     4381 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4441 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4501 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4561 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4621 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4681 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4741 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4801 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4861 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4921 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4981 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5041 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5101 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5161 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5221 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5281 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5341 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5401 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5461 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5521 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5581 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5641 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5701 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5761 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5821 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5881 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5941 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6001 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6061 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6121 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6181 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6241 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6301 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6361 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6421 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6481 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6541 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6601 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6661 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6721 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6781 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6841 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6901 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     6961 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     7021 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     7081 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     7141 cggccagtga attgtaatac gactcactat agggcgaatt g
//
