LOCUS       sequence-402675-        6030 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses split-intein fused MSPdH4-6 for circularization during 
            expression (internal His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6030)
  AUTHORS   Miehling J, Goricanec D, Hagn F
  TITLE     A Split-Intein-Based Method for the Efficient Production of 
            Circularized Nanodiscs for Structural Studies of Membrane Proteins.
  JOURNAL   Chembiochem. 2018 Sep 17;19(18):1927-1933. doi: 
            10.1002/cbic.201800345. Epub 2018 Jul 30.
  PUBMED    29947468
REFERENCE   2  (bases 1 to 6030)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1002/cbic.201800345"; journalName: "Chembiochem"; date: 
            "2018-09-17- 17"; volume: "19"; issue: "18"; pages: "1927-1933"
COMMENT     Sequence Label: pET28a-cMSPdH4-6
FEATURES             Location/Qualifiers
     source          1..6030
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(818..835)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             971..993
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1008..1032
                     /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)."
     primer_bind     complement(1032..1051)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1033..1051)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1135..1154)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1360..1437
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1438..2520
                     /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     complement(1457..1476)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3329..3520
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3514..3536)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3622..3761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3677..3696
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3776..3793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3947..4535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4027..4046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4646..4665)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4657..5472
                     /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             4657..5472
                     /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     complement(4730..4749)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5564..6019)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5701..5722)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5913..5932
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcaa ttcggcaggt tgtcaacgcg catcagatcc agttcacgtt
      181 cgaagatttc atcaatcggc agcatttgac cgtccaccgt cataaatttg tgatctttgg
      241 tggcgcggat cagagaacca tcttccagac aatattcaaa cacttcctgt tcgccacggt
      301 catgccattg cgcgaccggc tgcgtgtaaa tgttgccatt gttgtcaacg ctatacacgg
      361 tgcattcaat gcgtttttcc acaattttac caatcggcag cagaccgtat tccaccgtca
      421 ggatttccgt ttcataggac agacaaccct gggtgttcag ttttttggta tactcttcca
      481 gcgcgctcag aaagctcact ttaaagcttt ccagcaccgg cagcaggccc tgacgcagat
      541 cttccagcgc cggtttcgct ttttcgctca gggtgctcag atgttcggtc gctttcgcat
      601 gatattccgc cagacgcgca ccgccgtttt ctttcagcgc ttccagacgg gccgccagac
      661 gctgacgcag ttcatcgcta tacggctgca ctttcgcttt cacctcttcc agatctttgc
      721 tcatttcctg acgcaggcct tcggtttctt tttccaggtt atcccaaaat tcctgggtca
      781 ccgggcccag ctgttcacgc agtttgctaa aggtgctgtg atgatggtgg tgatgaccgt
      841 taaagcagtt ggatgcgata aagccatttt tcagggcaaa gttgtggtca cgttcgacac
      901 caatatcgta gacattctgt ttgcccagat atttacgggt cgcgatttta atgctgctgc
      961 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1021 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
     1081 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     1141 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     1201 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     1261 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     1321 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     1381 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     1441 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     1501 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     1561 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1621 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1681 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1741 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1801 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1861 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     1921 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     1981 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     2041 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     2101 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     2161 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     2221 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     2281 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     2341 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     2401 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     2461 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2521 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     2581 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2641 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     2701 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     2761 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     2821 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     2881 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     2941 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3001 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3061 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3121 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3181 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     3241 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     3301 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     3361 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     3421 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     3481 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3541 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3601 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3661 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
     3721 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     3781 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3841 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3901 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3961 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4021 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4081 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4141 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4201 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4261 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4321 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4381 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4441 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4501 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4561 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa
     4621 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     4681 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     4741 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     4801 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     4861 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     4921 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     4981 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     5041 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     5101 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     5161 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     5221 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     5281 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     5341 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     5401 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     5461 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5521 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5581 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5641 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5701 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5761 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5821 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5881 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5941 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     6001 gcgccgctac agggcgcgtc ccattcgcca
//
