LOCUS       sequence-463803-        6719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered SUMO protease expression plasmid designed for efficient 
            protease-mediated elution..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6719)
  TITLE     Molecular biology strategies for optimized expression and 
            purification using SUMO protease-mediated elution.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6719)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAIP1
FEATURES             Location/Qualifiers
     source          1..6719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..36
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1489..1506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1559..1577)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1573..1620
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1657..2112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1744..1763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1954..1975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2204..3019)
                     /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(2204..3019)
                     /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     2927..2946
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3011..3030
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3141..3729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3630..3649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3883..3900
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3915..4054
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3980..3999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4140..4162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4156..4347)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5156..6238)
                     /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     6200..6219
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6239..6316)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6522..6541
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6629..6648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6629..6647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6648..6672
                     /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             6687..6709
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgggacatc accaccatca tcaccaccat catcatagtg cagaggcaat gaatatgaat
       61 atgaatcaag acgccgtgtt catcccgccg cctgagggtg agcaatacga gagaaaggaa
      121 aagcaggaaa tccagcaaac tagttatctt caaagtcaag tgaaagtacc attggtgaat
      181 ttaccggcgc cattcttctc gacgtcattt tctgctcaag aaattctggg agagggcttt
      241 caagcctcta tctcccgcat aagcgctgta tcagaggagt taagtagcat tgagatacca
      301 gaactcgcag aggaagctcg aagagatttc gctgctaaga cccgagagca agaaatgctt
      361 agcgcaaatt accagaaaga ggtagaaaga aagacagagg cttaccgtaa acaacaggaa
      421 gttgaagccg ataagatacg taaggaatta gagaaacaac acttgcgcga tgttgaattt
      481 agaaaggaca tcgttgaaat ggccatagag aatcaaaaga agatgataga cgtggagtcc
      541 agatacgcca agaaggacat ggatagagag cgcgtaaagg tgagaatgat gttggaacaa
      601 cagaagttcc actctgatat tcaagttaac ctggacagct ctgcggctgg cacagagacg
      661 ggtggtcagg tagtaagcga gtcacaaaag ttcacggagc ggaaccgcca aataaagcaa
      721 tcagggttac gcccgccggc tcggcaactg attacacctt taagcgagga gtggcgcagt
      781 agagtcgaag cagctcggaa tgcaaatcct gcaactgagt tggccaagac cttagagggg
      841 caaccccttg tccgacgcga ttttgaagag aaattattgc cggcaacagc ttggctgaat
      901 gataatgtga ttattggggc catattttat atagctgatt atgtgaatac gaagaaggga
      961 gccccaaatc aagaacctaa gtgtacagcc tttacaagtt tcttttggcc ccgcttgtta
     1021 tcgcacggcc caggtggctg tggacgcctt ttacgtagag ctaatgtccg caaagcaaat
     1081 ttcttagaca tagatacaat actgatacct atctgtgagt catcccattg gacgcttgca
     1141 gtcatacgac ctggacgacg cacggtgtcc cacttggata gcatggcggc cggacgaggc
     1201 tcagaacggg taaaggccaa gcttttagag ttagtaaagt ttgtgcttga ggatcaattc
     1261 gtcgaggctg agtggcaagc agtcgatttt caagcgccgc gccagactaa tgggtgggac
     1321 tgtggggtat tcacaataac taatgccata tgcttagctt taggggtaga cccggctcag
     1381 gcatatactg aggcccagct tcccttacaa cgacaacgaa tagccgcagt attgttaaac
     1441 ggtggcttta agggcgactt tacactggat gatttgtgat gactcgagca ccaccaccac
     1501 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1561 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1621 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1681 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1741 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1801 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1861 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1921 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1981 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2041 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2101 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2161 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2221 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2281 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     2341 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     2401 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2461 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2521 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2581 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2641 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2701 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2761 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2821 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2881 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2941 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     3001 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     3061 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3121 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3181 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3241 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3301 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3361 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3421 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3481 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3541 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3601 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3661 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3721 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3781 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3841 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3901 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3961 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4021 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4081 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4141 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4201 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4261 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4321 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4381 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4441 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4501 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4561 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4621 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4681 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4741 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4801 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4861 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4921 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4981 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5041 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5101 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5161 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5221 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5281 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5341 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5401 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     5461 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5521 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5581 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5641 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5701 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5761 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5821 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5881 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5941 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6001 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6061 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6121 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6181 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6241 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6301 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6361 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6421 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6481 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6541 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     6601 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     6661 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatg
//
