LOCUS       sequence-66259-e        6093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein overexpression with the modified LacIWF repressor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6093)
  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 6093)
  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: pLIGFP_W220F
FEATURES             Location/Qualifiers
     source          1..6093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9..27
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    28..52
                     /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)."
     RBS             67..89
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(106..108,109..822)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(148..169)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(409..428)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     756..777
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             829..846
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(899..917)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      913..960
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      997..1452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1084..1103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1294..1315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1479..1583
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1584..1652,1653..2444)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1802..1821)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2615..3203
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3104..3123
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3357..3374
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3389..3531
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3457..3476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3617..3639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3633..3824)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4633..5715)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5677..5696
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5716..5793)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5999..6018
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
       61 aataattttg tttaacttta agaaggagat atacatatgg gcaaagtgag caagggcgag
      121 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      181 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      241 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
      301 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      361 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      421 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      481 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
      541 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
      601 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
      661 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
      721 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
      781 gccgccggga tcactctcgg catggacgag ctttacaagt aactcgagca ccaccaccac
      841 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      901 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      961 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1021 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1081 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1141 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1201 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1261 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1321 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1381 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1441 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1501 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1561 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1621 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1681 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1741 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1801 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     1861 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1921 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1981 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2041 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2101 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2161 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2221 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2281 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2341 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2401 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2461 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2521 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2581 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2641 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2701 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2761 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2821 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2881 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2941 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3001 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3061 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3121 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3181 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3241 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3301 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3361 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3421 gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata
     3481 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3541 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3601 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3661 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3721 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3781 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3841 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3901 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3961 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4021 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4081 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4141 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4201 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4261 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4321 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4381 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4441 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4501 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4561 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4621 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4681 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggtgctagcg tggtttttct
     4741 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4801 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4861 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4921 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4981 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5041 accggacatg gcactgaagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5101 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5161 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5221 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5281 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5341 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5401 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5461 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5521 gccaatcagc aagctttgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5581 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5641 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5701 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5761 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5821 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5881 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5941 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6001 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6061 tgcgtccggc gtagaggatc gagatctcga tcc
//
