LOCUS       sequence-65438-e        5080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overproduction of LacI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5080)
  AUTHORS   Gatti-Lafranconi P, Dijkman WP, Devenish SR, Hollfelder F
  TITLE     A single mutation in the core domain of the lac repressor reduces 
            leakiness.
  JOURNAL   Microb Cell Fact. 2013 Jul 8;12:67. doi: 10.1186/1475-2859-12-67.
  PUBMED    23834731
REFERENCE   2  (bases 1 to 5080)
  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: "Microb Cell
            Fact."; date: "2013-07-8"; volume: "12:67. doi"; pages: " 
            10.1186/1475-2859-12-67"
COMMENT     Sequence Label: pBAD-LacI
FEATURES             Location/Qualifiers
     source          1..5080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             128..1207
                     /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(147..166)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1214..1231
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1287..1304)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1457..1543
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1635..1662
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1681..1772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1773..1841,1842..2633)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1991..2010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2804..3392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3293..3312
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3546..3563
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3578..3718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3644..3663)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3804..3826
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3982..4860)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(4887..5080,1..91)
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
ORIGIN
        1 gtcacacttt gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc
       61 tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac aggaggaatt
      121 aaccatggtg aaaccagtaa cgctatacga tgtcgcagag tatgccggtg tctcttatca
      181 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
      241 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
      301 caaacaaagc ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
      361 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
      421 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
      481 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
      541 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
      601 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
      661 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
      721 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
      781 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      841 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      901 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
      961 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     1021 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     1081 gttgcccgtc tcactggtga aaagaaaaac cacgctagca cccaatacgc aaaccgcctc
     1141 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     1201 cgggcaggtc gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt
     1261 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     1321 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     1381 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     1441 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1501 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     1561 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     1621 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     1681 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1741 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1801 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1861 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1921 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1981 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2041 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2101 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2161 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2221 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2281 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2341 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2401 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2461 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2521 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2581 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2641 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2701 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2761 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2821 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2881 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2941 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3001 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3061 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3121 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3181 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3241 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3301 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3361 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3421 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3481 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3541 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3601 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     3661 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3721 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3781 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3841 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcataatg
     3901 tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg tcaagccgtc
     3961 aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc
     4021 acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa
     4081 atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt
     4141 gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat
     4201 ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc
     4261 gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc
     4321 gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag
     4381 taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc
     4441 ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg
     4501 aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg
     4561 cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg
     4621 atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc
     4681 ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc
     4741 cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc
     4801 caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat
     4861 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
     4921 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
     4981 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
     5041 taatcacggc agaaaagtcc acattgatta tttgcacggc
//
