LOCUS       sequence-471038-        6444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Superfolder GFP C-terminally fused to His-Smt3-tagged AesC split 
            intein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6444)
  AUTHORS   Humberg C, Yilmaz Z, Fitzian K, Dorner W, Kummel D, Mootz HD
  TITLE     A cysteine-less and ultra-fast split intein rationally engineered 
            from being aggregation-prone to highly efficient in protein 
            trans-splicing.
  JOURNAL   Nat Commun. 2025 Mar 19;16(1):2723. doi: 10.1038/s41467-025-57596-x.
  PUBMED    40108172
REFERENCE   2  (bases 1 to 6444)
  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: "Nat 
            Commun."; date: "2025-03-19"; volume: "16(1):2723. doi"; pages: " 
            10.1038/s41467-025-57596-x"
COMMENT     Sequence Label: pET28b_H6-Smt3-AesC-GFP
FEATURES             Location/Qualifiers
     source          1..6444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             157..450
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1309..1326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1379..1397)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1393..1440
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1477..1932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1564..1583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1774..1795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2024..2839)
                     /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(2024..2839)
                     /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     2747..2766
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2831..2850
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2961..3549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3450..3469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3703..3720
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3735..3874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3800..3819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3960..3982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3976..4167)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4976..6058)
                     /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     6020..6039
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6059..6136)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6342..6361
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatgt cggactcaga agtcaatcaa
      181 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
      241 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
      301 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
      361 ggtattagga ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
      421 attgaggctc acagagaaca gattggtgga tccatgatcg agttcatcga gttcgaaatt
      481 gaagatctgg gcgtgatgga aatcgatgtg tatgatattg aagtggatgg caaccacaac
      541 ttttttggca atgatattct ggtgcacaac agcgtgtatc tgaatggtac cagcaaaggc
      601 gaagaactgt ttaccggcgt ggtgccgatt ctggttgaac tggatggcga tgttaatggc
      661 cataagttta gcgtgcgcgg cgaaggcgaa ggcgatgcga ccaacggcaa actgaccctg
      721 aagtttattt gcaccaccgg caaactgccg gtgccgtggc cgaccctggt gaccaccctg
      781 acctatggcg tgcagtgctt tagccgctat ccggatcaca tgaaacgcca tgatttcttt
      841 aagtccgcga tgccggaagg ctatgttcag gaacgcacca ttagctttaa agatgatggc
      901 acctataaga cccgcgcgga agtgaaattt gaaggcgata ccctggtgaa ccgcattgaa
      961 ctgaaaggca ttgacttcaa agaagatggc aacattctgg gccataaact ggagtacaat
     1021 ttcaacagcc ataacgtgta tattaccgcg gacaaacaga agaacggcat caaagcgaat
     1081 ttcaaaatcc gccataatgt ggaagatggc agcgtgcagc tggcggatca ctatcagcag
     1141 aataccccga tcggcgatgg cccggtgctg ctgccggaca atcactacct gagcacccag
     1201 tccgtgctga gcaaagatcc gaatgagaaa cgcgatcaca tggttctgct ggagtttgtg
     1261 accgcggcgg gtatcactca tggttaaaag cttgcggccg cactcgagca ccaccaccac
     1321 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1381 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1441 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1501 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1561 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1621 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1681 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1741 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1801 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1861 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1921 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1981 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2041 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2101 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     2161 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     2221 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2281 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2341 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2401 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2461 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2521 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2581 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2641 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2701 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2761 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2821 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2881 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2941 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3001 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3061 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3121 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3181 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3241 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3301 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3361 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3421 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3481 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3541 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3601 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3661 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3721 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3781 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3841 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3901 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3961 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4021 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4081 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4141 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4201 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4261 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4321 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4381 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4441 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4501 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4561 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4621 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4681 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4741 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4801 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4861 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4921 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4981 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5041 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5101 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5161 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5221 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5281 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5341 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5401 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5461 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5521 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5581 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5641 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5701 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5761 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5821 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5881 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5941 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6001 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6061 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6121 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6181 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6241 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6301 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6361 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     6421 atcgagatct cgatcccgcg aaat
//
