LOCUS       sequence-39750-e        6222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of tetracycline-inducible CRE in Trypanosoma brucei.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6222)
  AUTHORS   Scahill MD, Pastar I, Cross GA
  TITLE     CRE recombinase-based positive-negative selection systems for 
            genetic manipulation in Trypanosoma brucei.
  JOURNAL   Mol Biochem Parasitol. 2008 Jan;157(1):73-82. Epub 2007 Oct 6.
  PUBMED    18006158
REFERENCE   2  (bases 1 to 6222)
  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: "Mol Biochem
            Parasitol."; date: "2008-01"; volume: "157(1)"; pages: "73-82. Epub 
            2007 Oct 6"
COMMENT     Sequence Label: pLEW100cre-EP1
FEATURES             Location/Qualifiers
     source          1..6222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1082..1456)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(1599..1618)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1600..1618)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1883..1901
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1904..1922
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2103..3134
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     promoter        complement(3458..3476)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3459..3476)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3767..3786)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3997..4016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4116..4138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4176..4194)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4262..4366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4367..4435,4436..5227)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHSMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4585..4604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5398..5986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5887..5906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6140..6157
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaattcgagc tcatatagtt ggtatgtatt ctaattccag actactggcg tggataaaca
       61 tgtcccctga ttaaaaggaa agattccata gcccattagt gcaaagataa ttggtacacc
      121 gtcaaaaaca catgggagac cacacgatac gacactgtga ccgtagcatc aattccgcac
      181 tcgatataac tctatcgaag aacttccatg gtacaaactg gtcgccacgg tctgctccac
      241 acggagatca tcgtatcatt tttatcgata gcggccgcta tcgatgtatg ccttggccct
      301 gatggcatgc caatttcact acaacggact atgtggaccc cgttatcatg gaaatgcgct
      361 agttggagga agttagaccg cgccggaaaa gagaggggta gagaaaatga caacttggaa
      421 gatatccaca cacgcacggt gaaacgttag caacaattat tagggaagca cgcttgccct
      481 agtcccacga gtaaccaaga ctccaaaagc ctttctggca cagagagcga gccgaaatgg
      541 aaaagagaaa caatgcctgc actaacacta ctgagcgatt cgcctcgccg cggaggaccg
      601 aatactaata acgacacttg cggtcaaaaa gtagaagaac aaatgctcaa cgatgagtga
      661 atcaggttag ggtagttgga aaattataca gaatgtcttt ggcaacacac cggcnnnctt
      721 gcatgcctgc aaggccttgc agaatactgc atagataaca aacgcatcaa acacaactgg
      781 tggcactatt tcaatcatgt cgacacacca agacgagaaa atttgagtta aacaagatgc
      841 ctacaaccct atatttatta tcttcaagtg atcttctctt ctcttgtgcg ctgtacgtaa
      901 atgtgttgca aataaggcat agggctgagt acaggcacca cacgcgagga aaatcataag
      961 cactagccgt gtcaactcac agcgactaca gaacaatttt ggccacacaa cccggtgtta
     1021 ggatctccga ggcctgggac ccgtgggccg ccgtcggacc ggcggtgttg gtcggcgtcg
     1081 gtcagtcctg ctcctcggcc acgaagtgca cgcagttgcc ggccgggtcg cgcagggcga
     1141 actcccgccc ccacggctgc tcgccgatct cggtcatggc cggcccggag gcgtcccgga
     1201 agttcgtgga cacgacctcc gaccactcgg cgtacagctc gtccaggccg cgcacccaca
     1261 cccaggccag ggtgttgtcc ggcaccacct ggtcctggac cgcgctgatg aacagggtca
     1321 cgtcgtcccg gaccacaccg gcgaagtcgt cctccacgaa gtcccgggag aacccgagcc
     1381 ggtcggtcca gaactcgacc gctccggcga cgtcgcgcgc ggtgagcacc ggaacggcac
     1441 tggtcaactt ggccatggtg atatagctta ttttatggca gcaacgagac cttacgtaaa
     1501 gcaaaactaa ataagagcgg agactgcaat gcagagtaaa aaaaaaaaaa acatgaattt
     1561 cagaagacct tgctgtgccc ccggtacggg ctagatctcc ctatagtgag tcgtattaat
     1621 caggtaccgt cattggggtt aagcggaaag gtgtgtgtca gtaggttgtg aggtgaaagc
     1681 gttttcagat gcatagtgag cttaatgtcc ttttcacagt atatcgtgtc tgataggtat
     1741 ctcttattag tatagtcgaa tactagtcaa tagtgcgttt tgtgcaaaat gtccattttg
     1801 tggcagtgat ggggttgttt tatgctattc cgtgtctctg ggtgggcgtg cattgaaaat
     1861 aggggttatc gggtgaggga tctccctatc agtgatagag atctccctat cagtgataga
     1921 gatccctgag tactgagttt aacatgttct cgtcccgggc tgcacgcgcc ttcgagtttt
     1981 ttttcctttt ccccattttt ttcaacttga agacttcaat tacaccaaaa agtaaaattc
     2041 acaagctcga ggggcagagc cgatcctgta cactttactt aaaaccatta tctgagtgtg
     2101 aaatgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga
     2161 gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc
     2221 atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata
     2281 accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg
     2341 cgcgcggtct ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc
     2401 gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga
     2461 tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca
     2521 ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta
     2581 atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga
     2641 tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa
     2701 cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac
     2761 tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt
     2821 gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg
     2881 ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg
     2941 gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg
     3001 cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata
     3061 ttgtcatgaa ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg
     3121 aagatggcga ttagccatta acgcggatcc tgcccattta gttggctttt cccttgtctc
     3181 gtgtcttttc cgtggaaagg ttcccggagt aatctgatgg cacagcaggg aggtgcgcct
     3241 gcagaatgcc ttattaacca tcgcctgaga cccacagccc tgtagatttc tgtgatgttt
     3301 cggttgcgta ttccataatt ttaagcgttt cacttctatt ttttttcatt cctttgaatt
     3361 tggatcttaa aattatcatt ggtgccttgt gttattgtgc gtgctgcgtg tgaatttggt
     3421 gctctgcctt tttgcaggca tgcaagctag cttgtattct atagtgtcac ctaaatcgta
     3481 tgtgtatgat acataaggtt atgtattaat tgtagccgcg ttctaacgac aatatgtaca
     3541 agcctaattg tgtagcatct ggcttactga agcagaccct atcatctctc tcgtaaactg
     3601 ccgtcagagt cggtttggtt ggacgaacct tctgagtttc tggtaacgcc gttccgcacc
     3661 ccggaaatgg tcagcgaacc aatcagcagg gtcatcgcta gccagatcct ctacgccgga
     3721 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     3781 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     3841 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcaccattcc
     3901 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     3961 cgcataaggg agagcgtcga tatggtgcac tctcagtaca atctgctctg atgccgcata
     4021 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4081 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4141 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4201 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4261 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4321 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4381 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4441 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4501 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4561 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4621 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4681 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4741 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4801 gctaaccgct tttttgcaca gcatggggga tcatgtaact cgccttgatc gttgggaacc
     4861 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4921 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4981 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5041 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5101 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5161 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5221 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5281 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5341 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5401 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5461 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5521 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5581 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5641 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5701 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5761 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5821 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5881 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5941 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6001 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6061 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6121 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     6181 caaaccgcct ctccccgcgc gttggccgat tcattaatgc ag
//
