LOCUS       sequence-422817-        4534 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression construct of AncSZ, an kinase domain engineered
            through ancestorial reconstruction of the kinase domains of both SYK
            and ZAP-70. For in vitro protein tyrosine phosphorylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4534)
  AUTHORS   Hobbs HT, Shah NH, Shoemaker SR, Amacher JF, Marqusee S, Kuriyan J
  TITLE     Saturation mutagenesis of a predicted ancestral Syk-family kinase.
  JOURNAL   Protein Sci. 2022 Oct;31(10):e4411. doi: 10.1002/pro.4411.
  PUBMED    36173161
REFERENCE   2  (bases 1 to 4534)
  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: "Protein 
            Sci."; date: "2022-10"; volume: "31(10):e4411. doi"; pages: " 
            10.1002/pro.4411"
COMMENT     Sequence Label: pET23a-H6-TEV-AncSZ
FEATURES             Location/Qualifiers
     source          1..4534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1674..1693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1674..1692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1724..1746
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1765..1782
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1804..1824
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2708..2725
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2778..2796)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2792..2839
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2876..3331
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2963..2982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3173..3194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3357..3461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3462..3530,3531..4322)
                     /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(3680..3699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4493..4534,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggccaggac ccaacgctgc ccgagatctc gatcccgcga aattaatacg
     1681 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga
     1741 aggagatata ccatgggcag cagccatcat catcatcatc acgattacga tatcccaacg
     1801 accgaaaacc tttacttcca gggccatatg gataaaaaac tgtatctgaa acgcgaacag
     1861 ctgatgctgg aagaaggcga actgggcagc ggcaactttg gcaccgtgaa aaaaggcgtg
     1921 tataaaatgc gcaaaaaaga aattccggtg gcggtgaaag tgctgaaaag cgaaaacgat
     1981 ccggcggtga aagatgaact gatgaaagaa gcggaattta tgcatcagct ggataacccg
     2041 tatattgtgc gcatgattgg catttgcgaa gcggaaagcc tgatgctggt gatggaactg
     2101 gcgccgctgg gcccgctgaa caaatttctg cagaaacata aagatcagat taccgtggaa
     2161 aacattgtgg aactgatgca tcaggtgagc atgggcatga aatatctgga agaaaaaaac
     2221 tttgtgcatc gcgatctggc ggcgcgcaac gtgctgctgg tgaaccagca ttatgcgaaa
     2281 attagcgatt ttggcctgag caaagcgctg ggcgcggatg ataactatta taaagcgaaa
     2341 accgcgggca aatggccgct gaaatggtat gcgccggaat gcattaactt tcataaattt
     2401 agcagcaaaa gcgatgtgtg gagctttggc gtgaccatgt gggaagcgtt tagctatggc
     2461 cagaaaccgt ataaaggcat gaaaggccag gaagtgctgc cgtttattga aaacggcgaa
     2521 cgcatggaat gcccggcgga atgcccggaa gaaatgtatg aactgatgaa agattgctgg
     2581 acctataaag cggatgatcg cccgggcttt gtggcggtgg aactgcgcct gcgcgattat
     2641 tattatgata ttagcaaata gggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc
     2701 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     2761 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2821 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2881 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2941 cttgccagcg ccatagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3001 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3061 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3121 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3181 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3241 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3301 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     3361 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3421 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3481 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3541 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3601 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3661 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3721 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3781 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3841 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3901 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3961 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     4021 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4081 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4141 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4201 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4261 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4321 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4381 ttaaaaggat ctaggtgaag atcctttttt ataatctcat gaccaaaatc ccttaacgtg
     4441 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4501 ctttttttct gcgcgtaatc tgctgcttgc aaac
//
