LOCUS       sequence-404713-        5644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO (Small Ubiquitin-Like Modifier) with 
            peptide tag for covalent bond formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5644)
  AUTHORS   Keeble AH, Wood DP, Howarth M
  TITLE     Design and Evolution of Enhanced Peptide-Peptide Ligation for 
            Modular Transglutaminase Assembly.
  JOURNAL   Bioconjug Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122.
  PUBMED    37289810
REFERENCE   2  (bases 1 to 5644)
  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: "Bioconjug 
            Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122."
COMMENT     Sequence Label: pET28a SUMO-DogTag2
FEATURES             Location/Qualifiers
     source          1..5644
                     /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(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(546..563)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             585..607
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    622..646
                     /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(646..665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(647..665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(749..768)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        974..1051
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1052..2134
                     /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(1071..1090)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2943..3134
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3128..3150)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3236..3375
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3291..3310
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3390..3407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3561..4149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3641..3660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4260..4279)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4271..5086
                     /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             4271..5086
                     /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(4344..4363)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5178..5633)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5315..5336)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5527..5546
                     /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 cggatctcag tggtggtggt ggtggtgctc gagtgcgtta gcaagcttat
      181 ttcggcggca gcggttcatt ggtgatataa tgtttaccat cggtaaattc atatgtagcc
      241 ggaatatcgc caccaccgat ctgttcgcga tgcgcttcaa taatatcgtt gtcttccata
      301 tccaggtctt ccggggtctg atcggcctga atacggatgc cgtcatacag aaagcgcagg
      361 ctatccattt ctttaccctg acgtttcgca aaggcttcca tcaggcgacg cagcggggtg
      421 gtttttttaa ttttaaaaaa gatttcgctg ctgccgtcgc taactttcag attaatatgg
      481 gtttccggtt tcacttccgg tttaacttcc ggtttcgctt cctggttcac ttcgctatcg
      541 ctcccgtgat gatgatggtg atggctccag ctacccatgg tatatctcct tcttaaagtt
      601 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
      661 tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
      721 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
      781 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
      841 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
      901 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
      961 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     1021 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1081 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1141 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1201 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1261 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     1321 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     1381 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     1441 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     1501 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     1561 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     1621 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     1681 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     1741 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     1801 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     1861 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     1921 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     1981 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2041 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2101 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2161 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     2221 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     2281 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     2341 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     2401 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     2461 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     2521 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     2581 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     2641 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     2701 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     2761 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     2821 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     2881 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     2941 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3001 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3061 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3121 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3181 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3241 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3301 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3361 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3421 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3481 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3541 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3601 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3661 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3721 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3781 ccggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3841 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3901 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3961 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4021 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4081 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4141 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4201 tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac
     4261 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     4321 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     4381 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     4441 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     4501 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     4561 caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     4621 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     4681 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     4741 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     4801 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     4861 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     4921 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     4981 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5041 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
     5101 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5161 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5221 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5281 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5341 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5401 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5461 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5521 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5581 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     5641 gcca
//
