LOCUS       sequence-11279-e        6188 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 6188)
  TITLE     George Cross Lab Plasmids
REFERENCE   2  (bases 1 to 6188)
  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"
COMMENT     Sequence Label: pLEW90
FEATURES             Location/Qualifiers
     source          1..6188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             674..1297
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(747..766)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1575..2600
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1575..2600
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(2948..2966)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2949..2966)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3257..3276)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3487..3506)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3606..3628
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3666..3684)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3752..3856
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3857..3925,3926..4717)
                     /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     complement(4075..4094)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4888..5476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5377..5396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5630..5647
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccgcatgt cgacgaagga ggttgatgag cagatgctga acgtgcagaa caagaactcg
       61 tcctacttca ttgagtggag atcccgaaca acatcaagtc ctctgtttgc gatatcccac
      121 ccaagggact caagatggct gtcaccttca ttggcaacaa cacctgcatc caggagatgt
      181 tccgccgtgt gggagagcag ttcaccctca tgttccgtcg caaggcgttc ttgcactggt
      241 acactggcga gggtatggac gagatggaat tcacggaggc agagtccaac atgaacgatc
      301 tcgtgtctga gtaccagcag taccaggatg ccacgattga ggaggagggc gagttcgacg
      361 aggaggagca atactagacg cggacggggc atttcccgtt cgtcattagc agtaggtaat
      421 gaagatgctc gagggtgctc aagctgtgta gcgcacgcgc cggtaatacg tctcactata
      481 gggagatcta ccggcgcgtt tccttacata tttctctaac aggcacggaa gcctaacaaa
      541 tacacttggc ttattttttt gccccctcat gtcttgtaca aatatttgcg atagcttagc
      601 tatcagccac attaatcaaa caagtatacc aacaagcccg aaaacagaaa ctcaactgca
      661 acgaagcttt catatgtcta gattagataa aagtaaagtg attaacagcg cattagagct
      721 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
      781 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
      841 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
      901 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
      961 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     1021 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     1081 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     1141 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     1201 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     1261 attagaaaaa caacttaaat gtgaaagtgg gtcttaagga tcctgcccat ttagttggct
     1321 tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga tggcacagca
     1381 gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag aaaatggggg
     1441 gattcgagcc cgggcacagc aaggtcttct gaaattcatg tttttttttt ttttactctg
     1501 cattgcagtc tccgctctta tttagttttg ctttacgtaa ggtctcgttg ctgccataaa
     1561 ataagctact agtgatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     1621 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     1681 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     1741 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     1801 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     1861 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     1921 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     1981 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     2041 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     2101 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     2161 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     2221 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     2281 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     2341 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     2401 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     2461 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     2521 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     2581 gtccgagggc aaaggaatag agtagatgcc gaccgggatc gatcccccga tcctaacacc
     2641 gggttgtgtg gccaaaattg ttctgtagtc gctgtgagtt gacacggcta gtgcttatga
     2701 ttttcctcgc gtgtggtgcc tgtactcagc cctatgcctt atttgcaaca catttacgta
     2761 cagcgcacaa gagaagagaa gatcacttga agataataaa tatagggttg taggcatctt
     2821 gtttaactca aattttctcg tcttggtgtg tcgacatgat tgaaatagtg ccaccagttg
     2881 tgtttgatgc gtttgttatc tatgcagtat tctgcaaggc cttgcaggca tgcaagctag
     2941 cttgtattct atagtgtcac ctaaatcgta tgtgtatgat acataaggtt atgtattaat
     3001 tgtagccgcg ttctaacgac aatatgtaca agcctaattg tgtagcatct ggcttactga
     3061 agcagaccct atcatctctc tcgtaaactg ccgtcagagt cggtttggtt ggacgaacct
     3121 tctgagtttc tggtaacgcc gttccgcacc ccggaaatgg tcagcgaacc aatcagcagg
     3181 gtcatcgcta gccagatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     3241 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     3301 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     3361 ctgttgggcg ccatctcctt gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     3421 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga tatggtgcac
     3481 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3541 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3601 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3661 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3721 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3781 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3841 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3901 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3961 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4021 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4081 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4141 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4201 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4261 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4321 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4381 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4441 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4501 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4561 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4621 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4681 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4741 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4801 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4861 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4921 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4981 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     5041 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5101 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5161 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5221 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5281 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5341 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5401 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5461 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5521 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5581 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5641 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5701 tcattaatgc agctgaccgt atcatgatga ctttctccat catcccatcc cccaaggtgt
     5761 ccgacactgt cgtcgagccg tacaatacga ctctctccgt gcaccaactt gtggaaaact
     5821 ccgatgagtc gatgtgcatt gacaacgagg cactgtacga tatttgcttc cgcaccctga
     5881 aactgacaac accaacgttc ggtgacctga accacttggt gtctgctgtt gtgtccggcg
     5941 tcacctgctg cctgcgcttc cctggtcagt tgaactctga cctccgtaag ttggctgtga
     6001 accttgtccc attcccgcgt ctgcacttct tcatgatggg cttcgccccg ctgaccagcc
     6061 gcggctcgca gcagtaccgc ggtctctccg tgcccgagct aacgcagcag atgttcgatg
     6121 cgaaaaacat gatgcaagct gcagatcctc gtcacggccg ctacctgaca gcgtctgcac
     6181 tcttccgc
//
