LOCUS       sequence-180024-        4600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of GFP with ""GLP"" N-tag and ""TRNGL"" for C-tag for
            cyclisation control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4600)
  AUTHORS   Mikula KM, Tascon I, Tommila JJ, Iwai H
  TITLE     Segmental isotopic labeling of a single-domain globular protein 
            without any refolding step by an asparaginyl endopeptidase.
  JOURNAL   FEBS Lett. 2017 Mar 30. doi: 10.1002/1873-3468.12640.
  PUBMED    28369872
REFERENCE   2  (bases 1 to 4600)
  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: "FEBS Lett. 
            2017 Mar 30. doi: 10.1002/1873-3468.12640."
COMMENT     Sequence Label: pITRSF4
FEATURES             Location/Qualifiers
     source          1..4600
                     /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)."
     CDS             88..381
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MGDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIETHREQIGG"
     CDS             391..408
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(439..467)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1062..1082
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1093..1110
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1154..1198
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1236..1254)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1250..1297
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1527..2342)
                     /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(1527..2342)
                     /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     2250..2269
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2343..2434)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2450..3199)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2460..2477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2700..2719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3395..4477)
                     /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     4439..4458
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4478..4555)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atatcatatg ggcgactcag aagtcaatca agaagctaag
      121 ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga
      181 tcttcagaaa tcttctttaa gatcaaaaag accactcctt taagaaggct gatggaagcg
      241 ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga
      301 attcaagctg atcagacccc tgaagatttg gacatggagg ataacgatat tattgagact
      361 cacagagaac agattggtgg tggattacca catcatcacc accatcactc tagaaaagga
      421 gaagaactct tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
      481 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcta catacggaaa actcaccctt
      541 aaatttattt gcactactgg aaaactacct gttccttggc caacacttgt cactactctg
      601 acctatggtg ttcaatgctt ttcccgttat ccggatcaca tgaaacggca tgactttttc
      661 aagagtgcca tgcccgaagg ttatgtacag gaacgcacta tatctttcaa agatgacggg
      721 aactacaaga cgcgtgctga agtcaagttt gaaggtgata cccttgttaa tcgtatcgag
      781 ttaaagggta ttgattttaa agaagatgga aacattctcg gacacaaact cgagtacaac
      841 tataactcac acaatgtata catcacggca gacaaacaaa agaatggaat caaagctaac
      901 ttcaaaattc gccacaacat tgaagatggt tccgttcaac tagcagacca ttatcaacaa
      961 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcgacacaa
     1021 tctgcccttt cgaaagatcc caacgaaaag cgtgaccaca tggtccttct tgagtttgta
     1081 actgctgctg ggttggtgcc acgcggtagt acccgaaatg gactgtaagc ttgcggccgc
     1141 actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     1201 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     1261 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa
     1321 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     1381 ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg
     1441 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1501 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1561 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1621 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1681 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1741 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1801 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1861 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta
     1921 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1981 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2041 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2101 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2161 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2221 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2281 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2341 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2401 tacatatttg aatgtattta gaaaaataaa caaataggca tgcagcgctc ttccgcttcc
     2461 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggtgtc agctcactca
     2521 aaagcggtaa tacggttatc cacagaatca ggggataaag ccggaaagaa catgtgagca
     2581 aaaagcaaag caccggaaga agccaacgcc gcaggcgttt ttccataggc tccgcccccc
     2641 tgacgagcat cacaaaaatc gacgctcaag ccagaggtgg cgaaacccga caggactata
     2701 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2761 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2821 acgctgttgg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2881 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2941 ggtaagacac gacttatcgc cactggcagc agccattggt aactgattta gaggactttg
     3001 tcttgaagtt atgcacctgt taaggctaaa ctgaaagaac agattttggt gagtgcggtc
     3061 ctccaaccca cttaccttgg ttcaaagagt tggtagctca gcgaaccttg agaaaaccac
     3121 cgttggtagc ggtggttttt ctttatttat gagatgatga atcaatcggt ctatcaagtc
     3181 aacgaacagc tattccgtta ctctagattt cagtgcaatt tatctcttca aatgtagcac
     3241 ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca
     3301 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3361 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3421 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3481 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3541 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3601 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3661 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3721 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3781 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3841 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     3901 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     3961 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4021 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4081 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4141 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4201 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4261 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4321 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4381 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4441 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4501 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4561 tctcgacgct ctcccttatg cgactcctgc attaggaaat
//
