LOCUS       sequence-196920-        6012 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 6012)
  TITLE     LIC N-terminal fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 6012)
  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: pET His6 GST TEV LIC cloning vector (1G)
FEATURES             Location/Qualifiers
     source          1..6012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..86
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     170..189
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        170..188
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    189..213
                     /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             228..250
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             269..286
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             296..949
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             959..979
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1045..1062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1115..1133)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1129..1176
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1213..1668
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1300..1319)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1510..1531
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1761..2576)
                     /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(1761..2576)
                     /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     2484..2503
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2568..2587
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2698..3286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3187..3206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3440..3457
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3472..3611
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3537..3556)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3697..3719
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3713..3904)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4713..5795)
                     /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     5757..5776
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5796..5873)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
       61 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
      121 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
      181 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      241 aagaaggaga tataccatgg gttcttctca ccatcaccat caccatggtt cttctatgtc
      301 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      361 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      421 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      481 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      541 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      601 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      661 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      721 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      781 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      841 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      901 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaag ggatcgagga
      961 aaacctgtac ttccaatcca atattggaag tggataacgg atccgaattc gagcgccgtc
     1021 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     1081 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1141 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1201 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1261 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1321 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1381 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1441 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1501 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1561 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1621 aacaaaaatt taacgcgaat tttaacaaac tagtaacgtt tacaatttca ggtggcactt
     1681 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1741 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1801 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     1861 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     1921 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     1981 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     2041 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     2101 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     2161 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaatg
     2221 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2281 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2341 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2401 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2461 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2521 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2581 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2641 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2701 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2761 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2821 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2881 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2941 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3001 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3061 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3121 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3181 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3241 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3301 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3361 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3421 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3481 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3541 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3601 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3661 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3721 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3781 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3841 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3901 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3961 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4021 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4081 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4141 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4201 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4261 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4321 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4381 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4441 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4501 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4561 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4621 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4681 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4861 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4921 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4981 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5041 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatacc ctcattcagc
     5101 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5161 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5221 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5281 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5341 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5401 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5461 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5521 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5581 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5641 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5701 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5761 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5821 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5881 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5941 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6001 ggccacgggg cc
//
