LOCUS       sequence-109759-        7000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eGFP in T. cruzi. The NheI-linearized form of this 
            plasmid could be efficiently integrated in to T. cruzi genome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7000)
  AUTHORS   Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL
  TITLE     CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in 
            Trypanosoma cruzi.
  JOURNAL   MBio. 2014 Dec 30;6(1). pii: e02097-14. doi: 10.1128/mBio.02097-14.
  PUBMED    25550322
REFERENCE   2  (bases 1 to 7000)
  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: "2014-12-30"; volume: "6(1). pii: e02097-14. doi"; pages: " 
            10.1128/mBio.02097-14"
COMMENT     Sequence Label: pTrex-n-eGFP
FEATURES             Location/Qualifiers
     source          1..7000
                     /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             join(1690..1692,1693..1695,1696..2409)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1690..1692,1693..1695,1696..2406)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1735..1756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1996..2015)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2343..2364
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3151..3954
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3214..3233)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3824..3843
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        complement(4815..4833)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4854..4870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4854..4870)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4867..4889)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4878..4894
                     /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(4902..4908,4909..4926,4927..4932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4947..4968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5085..5102)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5256..5844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5336..5355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6015..6806,6807..6875))
                     /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     6638..6657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6876..6980)
                     /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 tggagctcgg
      661 tgcaccctgg gatatttgcg cacccacctt tcccgtcgca atcggcgccg tccgaacgcg
      721 gaaatgttcc gagaataata cggctgggct gcgccgcgcg ttccccgcgt gaaccaacta
      781 ttgtgcgatg tgcagcatcc tattgtggtg ctttaccgca caccagtttc cttgcggggc
      841 ttatttggtg tgttggattt ttaatttttt gatgattcgg aggaattttt tgcaattttt
      901 gggtacccct aaaaaaattt tggagtttcc ggggttcgat ccaacttgtg cttttttaac
      961 cgtccatttg tgtctagtac atcatggagc ggtattctct ccggtggccc aggtggcgtc
     1021 ggaaggaatc ggtgtccctc aggatgtttg gtttttcgtt tttttttttg cggtcttttg
     1081 tttcgcatcg ccgtatcgtg tgcggcgcga ccttcttttt ggctcattgg tcagtgaggt
     1141 ttataataaa gtggatcatt tgggtttttg gccctttgcg gcgcacccgc ggaatggggg
     1201 agggattttc atataattgt agtgtacaca ttgtgtgtgg gtcgcaaaat gtcatgccgt
     1261 gtgcaatgtt tatttgcgtg ctgtgcgtgt ccttttgtga ttccaccact tgtgtgtatt
     1321 ttaccttatc gtctctccct gccgttggct gtgcgtgaat tgtgcttcgc attcgtggga
     1381 aatcaaaagg ggaatcaacg tgttcttttt ttttcagaag gggagtcaaa gcgccgtgtg
     1441 gatgccaagt ctgtgcaaga gttctagtgg atcccccaac gagtttcttc aaaatatgca
     1501 gcggattcac taagaaacat tttcacgcac gaaagcgaaa ttattatgat tgttattata
     1561 atactttttc tttgttgttt tatccactta ttattgttgt gttaaattgt ttttaccttt
     1621 tttcttttcc aacttctttt atgatgtctt ttcttttttt tttttgctct ataagttgtc
     1681 ttgtctagaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1741 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1801 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1861 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1921 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1981 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2041 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2101 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2161 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2221 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2281 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2341 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2401 tacaagtaac tcgagccatt tacgactcca aggcaacgct gcagaacaac ctgccgaagg
     2461 agcgccgctt cttcaagatt gtgtcgtggt acgacaacga gtggggatac tcccaccgcg
     2521 tggtggacct tgtacgccac atggcctcga aggatcgttc ggcaaggttg taggcgtggc
     2581 gatgacttca ggtctttctt ttgcgaatag ggatcttata atacacgatg cgtgtcccgt
     2641 gatgatcgtt accggtgctg ccacgatcca attgacacag cgtcaagagc aaaacaattt
     2701 tacttttccc tttaaggaca acaacaaaaa aatatataac ttttttttct tttttttttt
     2761 tttttgaaat tatatttatg gtcatctttg ggaaacaaaa agcagcaatt taatgatgcg
     2821 gaaggatgag tgaaataatg tttaatcaat gtacgaggat ttggggtatt gcaaggaaaa
     2881 tgtagatgat ttaattgggt gtgtgatgca gcttgtggta atttttgctc acttcccttt
     2941 ttgccacatc tttttagttt ttctgctttt ctttccccat tattccactt gtctctcttt
     3001 tcccacgttt cctgcacgaa tgcagaaagt gatattttta ctttgaaagc catctaccaa
     3061 caacaattac attgaacaga atttgggatt gcgaattaat tcctgcagcc ccgcgtgtgg
     3121 cctcgaacac cgagcgaccc tgcagccaat atgggatcgg ccattgaaca agatggattg
     3181 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3241 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3301 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3361 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3421 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3481 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3541 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3601 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3661 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3721 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3781 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3841 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3901 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggggat
     3961 cgatccgtgc acaggcgtta gccactctca ggattgccag caagtcttct gaatttgatc
     4021 tttggatgct cgggcagctc aaagaggaat cttagtcaca acctgccgaa ggagcgccgc
     4081 ttcttcaaga ttgtgtcgtg gtacgacaac gagtggggat actcccaccg cgtggtggac
     4141 cttgtacgcc acatggcctc gaaggatcgt tcggcaaggt tgtaggcgtg gcgatgactt
     4201 caggtctttc ttttgcgaat agggatctta taatacacga tgcgtgtccc gtgatgatcg
     4261 ttaccggtgc tgccacgatc caagtgacac agtgtcaaga gcaaaacaat tttacttttc
     4321 cctttaagga caacaacaaa aaaatatata actttttttt cttttttttt tttgaaatta
     4381 tatttatggt catctttggg aaacaaaaag cagcaattta atgatgcgga aggatgagtg
     4441 aaataatgtt taatcaatgt acgaggattt ggggtattgc aaggaaaatg tagatgattt
     4501 gattgggtgt atgatgcagc ttgtggtaat tttttacaac tttgcgttat tggaagattt
     4561 tggttgttca gtttcttttt gtttataact gactgtttta atttagtgtg attttttttt
     4621 tttaaggatt taagctgttt ttcttttttt ttctttttct tcactgatgg tgtttaaggt
     4681 gtagtttagt gtttgtgcat tatgcgtcgt agtgttgttg ttctttggac tttttcgaac
     4741 aatactgggc attatagccc acctgtgtat atgccgctag agtatgctag ccgtgtgggt
     4801 accagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
     4861 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4921 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4981 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5041 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5101 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5161 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5221 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5281 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5341 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5401 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5461 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5521 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5581 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5641 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5701 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5761 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5821 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5881 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5941 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6001 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6061 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6121 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6181 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6241 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6301 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6361 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6421 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6481 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6541 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6601 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6661 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6721 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6781 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6841 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6901 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6961 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
//
