LOCUS       sequence-362488-        5849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid contains LIC cassette upstream of folding reporter GFP 
            and TEV-cleavable 10x histidine purification tag. Compatible with T7
            promoter-based expression system in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5849)
  AUTHORS   Grave K, Bennett MD, Hogbom M
  TITLE     High-throughput strategy for identification of Mycobacterium 
            tuberculosis membrane protein expression conditions using folding 
            reporter GFP.
  JOURNAL   Protein Expr Purif. 2022 Oct;198:106132. doi: 
            10.1016/j.pep.2022.106132. Epub 2022 Jun 21.
  PUBMED    35750296
REFERENCE   2  (bases 1 to 5849)
  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: "Protein 
            Expr Purif."; date: "2022-10"; volume: "198:106132. doi"; pages: " 
            10.1016/j.pep.2022.106132. Epub 2022 Jun 21"
COMMENT     Sequence Label: pETcLIC-GFP
FEATURES             Location/Qualifiers
     source          1..5849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..65
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    80..219
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(145..164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     305..327
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(321..512)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1084..2166)
                     /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     2128..2147
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2167..2244)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2450..2469
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2557..2576
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2557..2575
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2576..2600
                     /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             2615..2637
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2684..3394
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2711..2739)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3334..3354
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3401..3430
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3453..3471)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3467..3514
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3551..4006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3638..3657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3848..3869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4121..4912,4913..4981))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4744..4763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4982..5074)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5155..5743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5644..5663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
       61 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      121 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      181 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
      241 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
      301 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
      361 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
      421 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
      481 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
      541 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
      601 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
      661 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
      721 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
      781 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
      841 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
      901 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
      961 acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     1021 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     1081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     1201 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     1261 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     1321 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
     1381 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     1441 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     1501 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     1561 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     1621 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     1681 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     1741 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     1801 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     1861 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     1921 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     1981 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     2041 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     2101 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     2161 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     2221 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     2281 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     2341 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     2401 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     2461 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     2521 cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat
     2581 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     2641 accatgggtg gtggatttaa atttatactt ccaaggtcaa ttcagcaaag gagaagaact
     2701 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     2761 ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aaactcaccc ttaaatttat
     2821 ttgcactact ggaaaactac ctgttccttg gccaacactt gtcactactc tgacctatgg
     2881 tgttcaatgc ttttcccgtt atccggatca catgaaacgg catgactttt tcaagagtgc
     2941 catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa
     3001 gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaggg
     3061 tattgatttt aaagaagatg gaaacattct cggacacaaa ctagagtaca actataactc
     3121 acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat
     3181 tcgccacaac attgaagatg gttccgttca actagcagac cattatcaac aaaatactcc
     3241 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct
     3301 ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc
     3361 tgggattaca catggcatgg atgagctcta caaaactagt catcatcatc accatcatca
     3421 tcaccatcat taattaacct aggctgctgc caccgctgag caataactag cataacccct
     3481 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     3541 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3601 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3661 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3721 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3781 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3841 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3901 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3961 caaaaattta acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg
     4021 gcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4081 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4141 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4201 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4261 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4321 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4381 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4441 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4501 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4561 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4621 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4681 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4741 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4801 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4861 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4921 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4981 tactcttcct ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga
     5041 tacatatttg aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg
     5101 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5161 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5221 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5281 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     5341 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5401 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5461 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5521 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5581 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5641 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5701 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5761 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5821 ccctgattct gtggataacc gtattaccg
//
