LOCUS       sequence-106345-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LIC E coli expression plasmid to produce TEV cleavable His tagged 
            iLOV fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  TITLE     B Smith Lab iLOF fusion plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7645)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pNIC28-Bsa4-iLOVf
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        28..46
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    47..71
                     /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             86..108
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             117..131
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             480..497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             522..542
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        558..1003
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1004..1090,1091..2425)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2533..2550
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2603..2621)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2617..2664
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2701..3156
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2788..2807)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2998..3019
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3249..4064)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3249..4064)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3972..3991
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4056..4075
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4186..4774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4675..4694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4928..4945
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4960..5102
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5028..5047)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5188..5210
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5204..5395)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6204..7286)
                     /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     7248..7267
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7287..7364)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7570..7589
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
       61 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacataatga
      121 atcacaaagt gatgatcggt accattgaga agaatttcgt aatcactgat ccacgtctgc
      181 cggacaaccc gatcatcttc gcttccgacg gcttcctgga gctgaccgag tattcccgtg
      241 aagagatcct gggccgtaac gcccgtttcc tgcagggccc ggagactgat caggcgacgg
      301 ttcagaaaat tcgcgatgct atccgtgacc agcgtgaaac tacggtgcaa ctgattaact
      361 acacgaaaag cggtaagaaa ttctggaacc tgctgcatct gcaaccagtc cgcgatcaga
      421 aaggcgagct gcagtatttc atcggcgttc aactggatgg ttctgaccac gtacatatgc
      481 accatcatca tcatcattct tctggtgtag atctgggtac cgagaacctg tacttccaat
      541 ccatggagac cgacgtccac atatacctgc cgttcactat tatttagtga aatgagatat
      601 tatgatattt tctgaattgt gattaaaaag gcaactttat gcccatgcaa cagaaactat
      661 aaaaaataca gagaatgaaa agaaacagat agatttttta gttctttagg cccgtagtct
      721 gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa
      781 acgagagtct aatagaatga ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc
      841 aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt tggcgtcacc ccttacatat
      901 tttaggtctt tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag
      961 aagcagaccg ctaacacagt acataaaaaa ggagacatga acgatgaaca tcaaaaagtt
     1021 tgcaaaacaa gcaacagtat taacctttac taccgcactg ctggcaggag gcgcaactca
     1081 agcgtttgcg aaagaaacga accaaaagcc atataaggaa acatacggca tttcccatat
     1141 tacacgccat gatatgctgc aaatccctga acagcaaaaa aatgaaaaat ataaagttcc
     1201 tgagttcgat tcgtccacaa ttaaaaatat ctcttctgca aaaggcctgg acgtttggga
     1261 cagctggcca ttacaaaaca ctgacggcac tgtcgcaaac tatcacggct accacatcgt
     1321 ctttgcatta gccggagatc ctaaaaatgc ggatgacaca tcgatttaca tgttctatca
     1381 aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag
     1441 cgacaaattc gatgcaaatg attctatcct aaaagaccaa acacaagaat ggtcaggttc
     1501 agccacattt acatctgacg gaaaaatccg tttattctac actgatttct ccggtaaaca
     1561 ttacggcaaa caaacactga caactgcaca agttaacgta tcagcatcag acagctcttt
     1621 gaacatcaac ggtgtagagg attataaatc aatctttgac ggtgacggaa aaacgtatca
     1681 aaatgtacag cagttcatcg atgaaggcaa ctacagctca ggcgacaacc atacgctgag
     1741 agatcctcac tacgtagaag ataaaggcca caaatactta gtatttgaag caaacactgg
     1801 aactgaagat ggctaccaag gcgaagaatc tttatttaac aaagcatact atggcaaaag
     1861 cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc
     1921 tgagttagca aacggcgctc tcggtatgat tgagctaaac gatgattaca cactgaaaaa
     1981 agtgatgaaa ccgctgattg catctaacac agtaacagat gaaattgaac gcgcgaacgt
     2041 ctttaaaatg aacggcaaat ggtacctgtt cactgactcc cgcggatcaa aaatgacgat
     2101 tgacggcatt acgtctaacg atatttacat gcttggttat gtttctaatt ctttaactgg
     2161 cccatacaag ccgctgaaca aaactggcct tgtgttaaaa atggatcttg atcctaacga
     2221 tgtaaccttt acttactcac acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat
     2281 tacaagctat atgacaaaca gaggattcta cgcagacaaa caatcaacgt ttgcgcctag
     2341 cttcctgctg aacatcaaag gcaagaaaac atctgttgtc aaagacagca tccttgaaca
     2401 aggacaatta acagttaaca aataaaaacg caaaagaaaa tgccgatatc ctattggcat
     2461 tgacggtctc cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg
     2521 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     2581 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     2641 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     2701 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2761 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2821 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2881 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2941 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3001 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3061 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3121 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     3181 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3241 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3301 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3361 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3421 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3481 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3541 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3601 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     3661 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3721 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     3781 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3841 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3901 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3961 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4021 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4081 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     4141 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4201 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4261 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4321 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4381 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4441 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4501 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4561 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4621 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4681 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4741 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4801 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4861 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4921 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4981 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     5041 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5101 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5161 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5221 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     5281 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     5341 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     5401 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     5461 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5521 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5581 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5641 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5701 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5761 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5821 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     5881 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5941 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     6001 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     6061 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     6121 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     6181 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6241 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     6301 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     6361 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     6421 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     6481 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     6541 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     6601 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6661 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     6721 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     6781 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     6841 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     6901 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     6961 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     7021 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     7081 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     7141 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     7201 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     7261 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     7321 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     7381 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     7441 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     7501 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     7561 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     7621 gccggccacg atgcgtccgg cgtag
//
