LOCUS       sequence-219875-        6355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for purification of Syk tSh2. Purified protein will have a snap tag 
            that can be conjugated to fluorescent dyes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6355)
  AUTHORS   Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, 
            Headley MB, Vale R
  TITLE     Chimeric antigen receptors that trigger phagocytosis.
  JOURNAL   Elife. 2018 Jun 4;7. pii: 36688. doi: 10.7554/eLife.36688.
  PUBMED    29862966
REFERENCE   2  (bases 1 to 6355)
  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: "Elife."; 
            date: "2018-06-4"; volume: "7. pii: 36688. doi"; pages: " 
            10.7554/eLife.36688"
COMMENT     Sequence Label: pGEX6-2 SNAP-Syk tSH2
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             951..1496
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(2405..2427)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2572..2590)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2658..2762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2763..2831,2832..3623)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2981..3000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3794..4382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4283..4302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4536..4553
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4626..4703
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4704..5786
                     /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(4723..4742)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5835..5840,5841..5858,5859..5865)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5873..5889
                     /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     5897..5913
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5897..5913
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5909..6355,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5929..5946)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5929..5945)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5938..5960)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atggacaaag
      961 attgcgaaat gaaacgtacc accctggata gcccgctggg caaactggaa ctgagcggct
     1021 gcgaacaggg cctgcatgaa attaaactgc tgggtaaagg caccagcgcg gccgatgcgg
     1081 ttgaagttcc ggccccggcc gccgtgctgg gtggtccgga accgctgatg caggcgaccg
     1141 cgtggctgaa cgcgtatttt catcagccgg aagcgattga agaatttccg gttccggcgc
     1201 tgcatcatcc ggtgtttcag caggagagct ttacccgtca ggtgctgtgg aaactgctga
     1261 aagtggttaa atttggcgaa gtgattagct atcagcagct ggcggccctg gcgggtaatc
     1321 cggcggccac cgccgccgtt aaaaccgcgc tgagcggtaa cccggtgccg attctgattc
     1381 cgtgccatcg tgtggttagc tctagcggtg cggttggcgg ttatgaaggt ggtctggcgg
     1441 tgaaagagtg gctgctggcc catgaaggtc atcgtctggg taaaccgggt ctgggaggta
     1501 gcggtagcgg tagcggtgga ggtggaagca gcactagaat ggcgggaagt gctgtggaca
     1561 gcgccaacca cctgacctac ttttttggca acatcacccg ggaagaggct gaagactacc
     1621 tggtccaggg aggcatgacc gatgggctct acctgctacg ccagagccgc aattacctgg
     1681 gtggttttgc tttgtcggtg gctcacaaca ggaaggcaca ccactacacc atcgagaggg
     1741 aacttaatgg cacctacgcc atctccgggg gcagggccca tgccagccca gcagacctct
     1801 gccattacca ctcccaggaa cctgatggcc ttatctgcct ccttaagaag cccttcaacc
     1861 ggcccccggg agtacagccc aagaccggac cctttgagga cctgaaggag aacctcatca
     1921 gggaatatgt gaaacagacc tggaaccttc agggccaggc tctggagcaa gccatcatca
     1981 gccagaagcc ccagctggag aagctgatcg ccaccacggc ccatgagaag atgccctggt
     2041 tccatggcaa catctccaga gatgaatcag agcagacggt cctcataggg tcaaagacca
     2101 atggaaaatt cctgatcagg gccagagaca acagcggctc ctatgctctg tgcctgctgc
     2161 acgaagggaa agtattgcac taccgcattg acagggacaa gaccgggaag ctctccattc
     2221 ctgaggggaa gaagtttgac accctctggc agctagtgga acattactct tacaagccag
     2281 atgggctact aagagtcctc acggtaccat gccaaaagat ttaagaattc ccgggtcgac
     2341 tcgagcggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg
     2401 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2461 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     2521 tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga
     2581 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2641 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2701 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2761 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2821 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2881 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2941 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3001 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3061 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3121 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3181 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3241 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3301 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3361 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3421 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3481 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3541 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3601 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3661 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3721 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3781 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3841 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3901 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3961 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4021 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4081 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4141 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4201 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4261 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4321 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4381 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4441 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4501 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4561 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     4621 attccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     4681 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     4741 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     4801 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     4861 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     4921 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     4981 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     5041 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     5101 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     5161 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     5221 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     5281 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     5341 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     5401 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     5461 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     5521 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     5581 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     5641 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacg ctagcgccca
     5701 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5761 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     5821 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5881 ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt
     5941 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     6001 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     6061 gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc
     6121 ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg
     6181 gcagatgcac ggttacgatg cgcccatcta caccaacgtg acctatccca ttacggtcaa
     6241 tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga
     6301 tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg gaatt
//
