LOCUS       sequence-491284-        6628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-SUMO-SPMc-SpyCatcher003-Ctag in bacterial cells for 
            calcium-induced NeissLock protein ligation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6628)
  AUTHORS   Lim SYT, Keeble AH, Howarth MR
  TITLE     Split NeissLock with Spy-Acceleration Arms Mammalian Proteins for 
            Anhydride-Mediated Cell Ligation.
  JOURNAL   ACS Chem Biol. 2025 Sep 15. doi: 10.1021/acschembio.5c00515.
  PUBMED    40947984
REFERENCE   2  (bases 1 to 6628)
  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: "ACS Chem 
            Biol. 2025 Sep 15. doi: 10.1021/acschembio.5c00515."
COMMENT     Sequence Label: His6-SUMO-SPMc-SpyCatcher003-Ctag
FEATURES             Location/Qualifiers
     source          1..6628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(60..1142)
                     /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     1104..1123
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1143..1220)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1426..1445
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1529..1548
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1529..1547
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1548..1572
                     /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             1587..1609
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1631..1648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2624..2962
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             3021..3038
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3091..3109)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3105..3152
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3189..3644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3276..3295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3486..3507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3736..4551)
                     /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(3736..4551)
                     /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     4459..4478
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4543..4562
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4673..5261
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5162..5181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5415..5432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5447..5586
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5512..5531)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5672..5694
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5688..5879)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
       61 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      121 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
      181 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
      241 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
      301 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
      361 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
      421 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
      481 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
      541 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
      601 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      661 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      721 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      781 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      841 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      901 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      961 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     1021 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     1081 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     1141 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     1201 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     1261 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     1321 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     1381 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     1441 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     1501 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1561 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
     1621 tagctggagc catcaccatc atcatcacgg gagcgatagc gaagtgaacc aggaagcgaa
     1681 accggaagtt aaaccggaag tgaaaccgga aacccatatt aatctgaaag ttagcgacgg
     1741 cagcagcgaa atctttttta aaattaaaaa aaccaccccg ctgcgtcgcc tgatggaagc
     1801 ctttgcgaaa cgtcagggta aagaaatgga tagcctgcgc tttctgtatg acggcatccg
     1861 tattcaggcc gatcagaccc cggaagacct ggatatggaa gacaacgata ttattgaagc
     1921 gcatcgcgaa cagatcggtg gtgcgttatt cgaccatcgc aaccaaggaa tccgtaccgc
     1981 gaccggctgg gtaagtgccg atgacggcct gcttgtacgc gatttgaatg gcaacggaat
     2041 tattgacaat ggagcagaac tttttggtga caatacgaag ttagctgacg ggtcattcgc
     2101 taaacatggc tatgcagcgc ttgccgagct ggatagtaat ggagacaaca ttatcaacgc
     2161 ggcagatgcc gcctttcaga cactgcgcgt ctggcaagat cttaaccaag acggtatttc
     2221 acaagccaac gaattacgca ctctggaaga gttaggtatt caatcgttgg atcttgcgta
     2281 taaagacgtg aacaagaatt tagggaacgg caacaccttg gctcaacagg gcagctacac
     2341 aaagacagac ggtacgacgg ccaagatggg agacctgctg ttggccgctg ataacttgca
     2401 cagccgtttc aaagataaag tcgaacttac cgcagaacag gccaaggccg cgaatcttgc
     2461 cggtatcggc cgtttacgcg acctgcgtga ggctgctgcc ctgtctgggg acttagcgaa
     2521 tatgttaaag gcatatagtg cggctgagac aaaggaagcc cagttagcgt tgttagacaa
     2581 tttaatccat aaatgggcag agacagacgg gagcggtgcc atggtaacca ccttatcagg
     2641 tttatcaggt gagcaaggtc cgtccggtga tatgacaact gaagaagata gtgctaccca
     2701 tattaaattc tcaaaacgtg atgaggacgg ccgtgagtta gctggtgcaa ctatggagtt
     2761 gcgtgattca tctggtaaaa ctattagtac atggatttca gatggacatg tgaaggattt
     2821 ctacctgtat ccaggaaaat atacatttgt cgaaaccgca gcaccagacg gttatgaggt
     2881 agcaactcca attgaattta cagttaatga ggacggtcag gttactgtag atggtgaagc
     2941 aactgaaggt gacgctcata ctggatccag tggtagcggg agcggtgagc cagaggctta
     3001 agcttgctaa cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     3061 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     3121 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     3181 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3241 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3301 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3361 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3421 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3481 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3541 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3601 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     3661 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3721 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     3781 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     3841 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     3901 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     3961 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     4021 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     4081 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     4141 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     4201 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     4261 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     4321 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     4381 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     4441 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     4501 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     4561 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     4621 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4681 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4741 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4801 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4861 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4921 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4981 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5041 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5101 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5161 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5221 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5281 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5341 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5401 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     5461 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     5521 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5581 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5641 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5701 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5761 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5821 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5881 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5941 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     6001 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     6061 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     6121 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     6181 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     6241 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cggtaaggca accccgccag
     6301 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     6361 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     6421 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     6481 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     6541 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     6601 gagctgactg ggttgaaggc tctcaagg
//
