LOCUS       sequence-251770-        3968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for recombinant expression in bacteria of SpyLigase. The 
            Howarth lab has created two improved peptide ligation enzymes. See 
            depositor comments below!.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3968)
  AUTHORS   Fierer JO, Veggiani G, Howarth M
  TITLE     SpyLigase peptide-peptide ligation polymerizes affibodies to enhance
            magnetic cancer cell capture.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Mar 17.
  PUBMED    24639550
REFERENCE   2  (bases 1 to 3968)
  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: "Proc Natl 
            Acad Sci U S A. 2014 Mar 17."
COMMENT     Sequence Label: SpyLigase
FEATURES             Location/Qualifiers
     source          1..3968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             115..137
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             157..174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             533..550
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(603..621)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      617..664
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      701..1156
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(788..807)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     998..1019
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1182..1286
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1287..1355,1356..2147)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1505..1524)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2318..2906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2807..2826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3060..3077
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3092..3231
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3157..3176)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3317..3339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3333..3524)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatct cgatcccgcg
       61 aaattaatac gactcactat agggagacca caacggtttc cctctagaaa taattttgtt
      121 taactttaag aaggagatat acatatgtcg tactaccatc accatcacca tcacgattac
      181 gacggtcagt ccggtgacgg caaagagtta gctggtgcaa ctatggagtt gcgtgattca
      241 tctggtaaaa ctattagtac atggatttca gatggacaag tgaaagattt ctacctgtat
      301 ccaggaaaat atacatttgt cgaaaccgca gcaccagacg gttatgaggt agcaactgct
      361 attaccttta cagttaatga gcaaggtcag gttactgtaa atggcaaagc aactaaaggt
      421 gggagtggtg gcagcggagg tagtggcgag gacagcgcta cccatattta aatggttgat
      481 gcttgaggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
      541 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
      601 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
      661 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
      721 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      781 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      841 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      901 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      961 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1021 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1081 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1141 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1201 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tggtaaatgc
     1261 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1321 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1381 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1441 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1501 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1561 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1621 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1681 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1741 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1801 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     1861 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1921 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1981 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2041 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2101 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2161 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2221 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2281 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2341 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2401 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2461 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2521 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2581 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2641 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2701 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2761 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2821 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2881 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2941 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3001 accgtattac cgcctttgag tgagctgata ccgatcgccg cagccgaacg accgagcgca
     3061 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3121 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3181 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3241 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3301 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3361 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3421 gcctgttcat ccgcgtccag ctcgttgagc ttctccagaa gcgttaatgt ctggcttctg
     3481 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3541 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3601 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3661 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3721 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3781 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3841 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3901 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3961 aacgacag
//
