LOCUS       sequence-433100-        6129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AavLEA1 expression (in BL21 DE3 E. coli strain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6129)
  AUTHORS   Tripathi R, Benz N, Culleton B, Trouve P, Ferec C
  TITLE     Biophysical Characterisation of Calumenin as a Charged F508del-CFTR 
            Folding Modulator.
  JOURNAL   PLoS One. 2014 Aug 13;9(8):e104970. doi: 
            10.1371/journal.pone.0104970. eCollection 2014.
  PUBMED    25120007
REFERENCE   2  (bases 1 to 6129)
  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: "PLoS One.";
            date: "2014-08-13"; volume: "9(8):e104970. doi"; pages: " 
            10.1371/journal.pone.0104970. eCollection 2014"
COMMENT     Sequence Label: pET15b-AavLEA1
FEATURES             Location/Qualifiers
     source          1..6129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(760..777)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(787..804)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             823..845
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    860..884
                     /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)."
     primer_bind     complement(884..903)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(885..903)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(987..1006)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1212..1289
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1290..2372
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1309..1328)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3684..3875
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3869..3891)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3977..4116
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4032..4051
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4131..4148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4302..4890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4382..4401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5061..5852,5853..5921))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5684..5703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5922..6026)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6094..6112
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atccttagtc gcggcccttg accttctcgc cagcctggtg
      361 gagcttctcg gcggcagtgt cgcgaagctc ctcggccttg cccttgccct gctgcttgta
      421 ctcctccatc ttctggcccg cgttctcgcg catttcgcca gcccgctgct tggtctcatc
      481 catcttctcc ccggcacgct gcttcgtctc ctctgccttc tcgccagcgc gctccttgaa
      541 ctcctgaccc ttctcctgcg cctgtccggt caggtcacgg atggtctcac cggcggaatc
      601 gcgaaactca gccgtcttct cggcggccgc ttgagccgtg ctcgagaggg tttccttcgt
      661 gctctcccat gcgttgacga ccttctcctt ggccgcctcc atgtagccct gctcctgctg
      721 ctcaccctgt cggttctggt tctgctgaga ggacatatgg ctgccgcgcg gcaccaggcc
      781 gctgctgtga tgatgatgat gatggctgct gcccatggta tatctccttc ttaaagttaa
      841 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
      901 ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
      961 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     1021 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     1081 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     1141 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1201 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1261 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1321 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1381 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1441 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1501 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1561 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1621 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1681 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1741 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1801 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1861 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1921 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1981 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     2041 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     2101 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2161 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2221 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2281 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2341 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2401 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2461 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2521 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     2581 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     2641 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     2701 ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg
     2761 gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc
     2821 atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct
     2881 cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta tgccgcctcg
     2941 gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc
     3001 cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc
     3061 acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg
     3121 tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc
     3181 gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc
     3241 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     3301 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     3361 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     3421 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     3481 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3541 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3601 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3661 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3721 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3781 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3841 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3901 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3961 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     4021 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     4081 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     4141 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4201 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4261 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4321 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4381 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4441 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4501 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4561 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4621 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4681 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4741 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4801 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4861 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4921 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4981 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5041 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5101 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5161 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5221 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5281 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5341 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     5401 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5461 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5521 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5581 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5641 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     5701 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5761 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5821 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5881 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5941 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6001 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6061 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     6121 cttcaagaa
//
