LOCUS       sequence-39749-e        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  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 6220)
  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-EP1-6G
FEATURES             Location/Qualifiers
     source          1..6220
                     /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(1595..1614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1596..1614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1879..1897
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1900..1918
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2100..3131
                     /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(3456..3474)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3457..3474)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3765..3784)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3995..4014)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4114..4136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4174..4192)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4260..4364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4365..4433,4434..5225)
                     /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(4583..4602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5396..5984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5885..5904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6138..6155
                     /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 atagcttatt ttatggcagc aacgagacct tacgtaaagc
     1501 aaaactaaat aagagcggag actgcaatgc agagtaaaaa aaaaaaaaac atgaatttca
     1561 gaagaccttg ctgtgccccc ggtacgggag atctccctat agtgagtcgt attaatcagg
     1621 taccgtcatt ggggttaagc ggaaaggtgt gtgtcagtag gttgtgaggt gaaagcgttt
     1681 tcagatgcat agtgagctta atgtcctttt cacagtatat cgtgtctgat aggtatctct
     1741 tattagtata gtcgaatact agtcaatagt gcgttttgtg caaaatgtcc attttgtggc
     1801 agtgatgggg ttgttttatg ctattccgtg tctctgggtg ggcgtgcatt gaaaataggg
     1861 gttatcgggt gagggatctc cctatcagtg atagagatct ccctatcagt gatagagatc
     1921 cctgagtact gagtttaaca tgttctcgtc ccgggctgca cgcgccttcg agttttggtt
     1981 gccttttgcc catttggttc aacttgaaga cttcaattac accaaaaagt aaaattcaca
     2041 agctcgaggg gcagagccga tcctgtacac tttacttaaa accattatct gagtgtgaaa
     2101 tgtccaattt actgaccgta caccaaaatt tgcctgcatt accggtcgat gcaacgagtg
     2161 atgaggttcg caagaacctg atggacatgt tcagggatcg ccaggcgttt tctgagcata
     2221 cctggaaaat gcttctgtcc gtttgccggt cgtgggcggc atggtgcaag ttgaataacc
     2281 ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta tcttctatat cttcaggcgc
     2341 gcggtctggc agtaaaaact atccagcaac atttgggcca gctaaacatg cttcatcgtc
     2401 ggtccgggct gccacgacca agtgacagca atgctgtttc actggttatg cggcggatcc
     2461 gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg
     2521 atttcgacca ggttcgttca ctcatggaaa atagcgatcg ctgccaggat atacgtaatc
     2581 tggcatttct ggggattgct tataacaccc tgttacgtat agccgaaatt gccaggatca
     2641 gggttaaaga tatctcacgt actgacggtg ggagaatgtt aatccatatt ggcagaacga
     2701 aaacgctggt tagcaccgca ggtgtagaga aggcacttag cctgggggta actaaactgg
     2761 tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc gaataactac ctgttttgcc
     2821 gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag ccagctatca actcgcgccc
     2881 tggaagggat ttttgaagca actcatcgat tgatttacgg cgctaaggat gactctggtc
     2941 agagatacct ggcctggtct ggacacagtg cccgtgtcgg agccgcgcga gatatggccc
     3001 gcgctggagt ttcaataccg gagatcatgc aagctggtgg ctggaccaat gtaaatattg
     3061 tcatgaacta tatccgtaac ctggatagtg aaacaggggc aatggtgcgc ctgctggaag
     3121 atggcgatta gccattaacg cggatcctgc ccatttagtt ggcttttccc ttgtctcgtg
     3181 tcttttccgt ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca
     3241 gaatgcctta ttaaccatcg cctgagaccc acagccctgt agatttctgt gatgtttcgg
     3301 ttgcgtattc cataatttta agcgtttcac ttctattttt tttcattcct ttgaatttgg
     3361 atcttaaaat tatcattggt gccttgtgtt attgtgcgtg ctgcgtgtga atttggtgct
     3421 ctgccttttc tgcaggcatg caagctagct tgtattctat agtgtcacct aaatcgtatg
     3481 tgtatgatac ataaggttat gtattaattg tagccgcgtt ctaacgacaa tatgtacaag
     3541 cctaattgtg tagcatctgg cttactgaag cagaccctat catctctctc gtaaactgcc
     3601 gtcagagtcg gtttggttgg acgaaccttc tgagtttctg gtaacgccgt tccgcacccc
     3661 ggaaatggtc agcgaaccaa tcagcagggt catcgctagc cagatcctct acgccggacg
     3721 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     3781 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     3841 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc accattcctt
     3901 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     3961 cataagggag agcgtcgata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4021 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4081 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4141 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4201 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4381 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4441 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4501 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4561 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4621 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4681 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4741 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4801 taaccgcttt tttgcacagc atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4861 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4921 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4981 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5041 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5101 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5161 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5221 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5521 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     5581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6061 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6121 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6181 aaccgcctct ccccgcgcgt tggccgattc attaatgcag
//
