LOCUS       sequence-491279-        5824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AviTag-His6-OAZ-SPM(FrpA 298-318)-GSG-SpyTag003 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 5824)
  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 5824)
  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: AviTag-His6-OAZ-SPM(FrpA 298-318)-GSG-SpyTag003
FEATURES             Location/Qualifiers
     source          1..5824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..116
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(136..213)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     419..438
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     522..541
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        522..540
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    541..565
                     /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             580..602
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             618..662
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             672..689
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1140..1187
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             1210..1227
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1280..1298)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1294..1341
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1378..1833
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1465..1484)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1675..1696
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1925..2740)
                     /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(1925..2740)
                     /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     2648..2667
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2732..2751
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2862..3450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3351..3370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3604..3621
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3636..3775
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3701..3720)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3861..3883
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3877..4068)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(4877..5824,1..135))
                     /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"
ORIGIN
        1 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
       61 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
      121 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
      181 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
      241 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
      301 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
      361 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      421 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      481 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
      541 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
      601 gatataccat gggcagcggc ctgaacgata tttttgaagc gcagaaaatt gaatggcatg
      661 aaggcagcag ccatcatcat catcatcact tctactcgga cgatcgtttg aatgtgaccg
      721 aagagttgac gtccaacgac aaaacacgta tcttgaatgt ccagtcccgt ctgactgacg
      781 ccaaacgtat taattggcgc actgtactta gcggtggcag tctttacatc gaaatcccag
      841 ggggcgcctt gcctgaaggc tctaaagatt cgtttgccgt gttgttggag tttgccgagg
      901 agcagttacg cgcagatcat gtgttcatcg ccttccacaa gaaccgcgaa gatcgtgcag
      961 cccttctgcg tactttctcc ttccttggct tcgagatcgt gcgtccaggg catccgctgg
     1021 tgcccaagcg tcctgacgct tgttttatgg catatacttt cgaaggcgta tatgatccgc
     1081 tggcgttgga cctggatggt gacggaatcg agacggtggc tgccaaagga gggagcggtc
     1141 gaggtgttcc acatattgtt atggttgatg cttacaagcg atacaagtaa gcttgctaac
     1201 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1261 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1321 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1381 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1441 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1501 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1561 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1621 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1681 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1741 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1801 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1861 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1921 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1981 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     2041 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     2101 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     2161 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2221 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2281 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2341 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     2401 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     2461 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2521 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2581 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     2641 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2701 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     2761 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     2821 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2881 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2941 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3001 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3061 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3121 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3181 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3241 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3301 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3361 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3421 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3481 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3541 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3601 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3661 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3721 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3781 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3841 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3901 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3961 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4021 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4081 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4141 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4201 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4261 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4321 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4381 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4441 ctcgcgtatc ggtgattcat tctgctaacc ggtaaggcaa ccccgccagc ctagccgggt
     4501 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4561 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4621 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4681 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4741 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4801 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4861 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4981 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5041 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5101 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5161 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5221 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5281 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5341 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5401 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5461 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5521 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5581 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5641 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5701 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5761 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5821 aatt
//
