LOCUS       sequence-197189-        6072 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 6072)
  TITLE     LIC N-terminal fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 6072)
  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 GFP TEV LIC cloning vector (1GFP)
FEATURES             Location/Qualifiers
     source          1..6072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     303..322
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     556..573
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    588..727
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(653..672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     813..835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(829..1020)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1829..2911)
                     /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     2873..2892
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2912..2989)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3195..3214
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3298..3317
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3298..3316
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3317..3341
                     /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             3356..3378
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3397..3414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(3424..3426,3427..4137)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3466..3487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4074..4095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4147..4167
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4233..4250
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4303..4321)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4317..4364
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4401..4856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4488..4507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4698..4719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4949..5764)
                     /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(4949..5764)
                     /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     5672..5691
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5756..5775
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5886..6072,1..402)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
       61 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      121 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      181 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      241 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      301 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      361 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      421 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      481 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      541 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
      601 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
      661 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
      721 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      781 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      841 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      901 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      961 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     1021 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     1081 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     1141 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     1201 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     1261 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     1321 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     1381 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     1441 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     1501 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     1561 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     1621 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     1681 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     1741 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     1801 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1861 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1921 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1981 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2041 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2101 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2161 gcgccatctg atcgttggca accagcatcg cagtgggaac gataccctca ttcagcattt
     2221 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2281 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     2341 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2401 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     2461 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2521 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2581 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2641 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2701 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2761 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2821 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2881 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2941 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     3001 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     3061 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     3121 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     3181 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     3241 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     3301 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     3361 ttaactttaa gaaggagata taccatgggt tcttctcacc atcaccatca ccatggttct
     3421 tctgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3481 ggcgacgtaa acggccacaa gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac
     3541 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3601 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3661 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc
     3721 ttcaaggacg acggcaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3781 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3841 aagctggagt acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac
     3901 ggcatcaagg cgaacttcaa gatccgccac aacgtcgagg acggcagcgt gcagctcgcc
     3961 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4021 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4081 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggg
     4141 atcgaggaaa acctgtactt ccaatccaat attggaagtg gataacggat ccgaattcga
     4201 gcgccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     4261 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     4321 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     4381 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4441 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4501 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4561 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4621 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4681 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4741 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4801 gctgatttaa caaaaattta acgcgaattt taacaaacta gtaacgttta caatttcagg
     4861 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4921 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     4981 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     5041 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     5101 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     5161 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     5221 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     5281 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     5341 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     5401 caacaatgtt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     5461 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     5521 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     5581 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     5641 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     5701 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     5761 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     5821 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5881 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5941 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6001 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6061 cacttcaaga ac
//
