LOCUS       sequence-174751-        6082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET LIC cloning vector for Biotin-His6-tev-yORF-mClover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6082)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 6082)
  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 mClover LIC cloning vector (H6-mClover)
FEATURES             Location/Qualifiers
     source          1..6082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     241..262
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             323..340
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(393..411)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      407..454
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      491..946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(578..597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     788..809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1039..1854)
                     /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(1039..1854)
                     /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     1762..1781
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1846..1865
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1976..2564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2465..2484
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2718..2735
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2750..2889
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2815..2834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2975..2997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2991..3182)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3991..5073)
                     /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     5035..5054
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5074..5151)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5357..5376
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5460..5479
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5460..5478
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5479..5503
                     /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             5518..5540
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5553..5597
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5598..5615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5616..5636
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(5673..5675,5676..6082,1..304)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(5715..5736)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca
       61 gaagaacggc atcaaggcta acttcaagat ccgccacaac gttgaggacg gcagcgtgca
      121 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
      181 caaccactac ctgagccatc agtccgccct gagcaaagac cccaacgaga agcgcgatca
      241 catggtcctg ctggagttcg tgaccgccgc cgggattaca catggcatgg acgagctgta
      301 caagtaagcg gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
      361 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      421 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
      481 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      541 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      601 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      661 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
      721 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      781 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
      841 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
      901 caaaaattta acgcgaattt taacaaacta gtaacgttta caatttcagg tggcactttt
      961 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1021 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1081 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1141 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1201 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1261 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1321 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1381 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     1441 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatgtt
     1501 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1561 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1621 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1681 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1741 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1801 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1861 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1921 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1981 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2041 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2101 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2161 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2221 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2281 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2341 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2401 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2461 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2521 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2581 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2641 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2701 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2761 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2821 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2881 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2941 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3001 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3061 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3121 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3181 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3241 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3301 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3361 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3421 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3481 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3541 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3601 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3661 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3721 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3781 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3841 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3901 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3961 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4021 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4081 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4141 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4201 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4261 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4321 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgataccct cattcagcat
     4381 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4441 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4501 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4561 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4621 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4681 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4741 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4801 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4861 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4921 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4981 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5041 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5101 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5161 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5221 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5281 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5341 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5401 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     5461 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     5521 gtttaacttt aagaaggaga tataccatgg gaggcctgaa cgatattttt gaagcgcaga
     5581 aaattgaatg gcatgaacac catcaccatc accatgaaaa cctgtacttc caatccaata
     5641 ttggtagtgg gagcaacggc agcagcggat ccgtgagcaa gggcgaggag ctgttcaccg
     5701 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc
     5761 gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca
     5821 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcgtggcct
     5881 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     5941 aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg
     6001 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     6061 tcaaggagga cggcaacatc ct
//
