LOCUS       sequence-251783-        6856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Shark tandem SH2 domains SNAP tagged for use as FRET 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6856)
  AUTHORS   Williamson AP, Vale RD
  TITLE     Spatial control of Draper receptor signaling initiates apoptotic 
            cell engulfment.
  JOURNAL   J Cell Biol. 2018 Nov 5;217(11):3977-3992. doi: 
            10.1083/jcb.201711175. Epub 2018 Aug 23.
  PUBMED    30139739
REFERENCE   2  (bases 1 to 6856)
  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"; doi: 
            "10.1083/jcb.201711175"; journalName: "J Cell Biol"; date: 
            "2018-11-5- 5"; volume: "217"; issue: "11"; pages: "3977-3992"
COMMENT     Sequence Label: pGEX Shark-tSH2
FEATURES             Location/Qualifiers
     source          1..6856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(65..70,71..86,87..93)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    101..117
                     /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             140..793
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     751..773
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             800..823
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             833..1378
                     /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(2788..2810)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2955..2973)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3041..3145
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3146..3214,3215..4006)
                     /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(3364..3383)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4177..4765
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4666..4685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4919..4936
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5009..5086
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5087..6169
                     /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(5106..5125)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6218..6223,6224..6241,6242..6248)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6256..6272
                     /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     6280..6296
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6280..6296
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6292..6819
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6312..6329)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6312..6328)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6321..6343)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga
       61 gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt
      121 cacacaggaa acagtattca tgtcccctat actaggttat tggaaaatta agggccttgt
      181 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
      241 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
      301 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
      361 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
      421 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
      481 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
      541 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
      601 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
      661 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
      721 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
      781 ccatcctcca aaatcggatc tggaagttct gttccagggg cccctgggat ccatggacaa
      841 agattgcgaa atgaaacgta ccaccctgga tagcccgctg ggcaaactgg aactgagcgg
      901 ctgcgaacag ggcctgcatg aaattaaact gctgggtaaa ggcaccagcg cggccgatgc
      961 ggttgaagtt ccggccccgg ccgccgtgct gggtggtccg gaaccgctga tgcaggcgac
     1021 cgcgtggctg aacgcgtatt ttcatcagcc ggaagcgatt gaagaatttc cggttccggc
     1081 gctgcatcat ccggtgtttc agcaggagag ctttacccgt caggtgctgt ggaaactgct
     1141 gaaagtggtt aaatttggcg aagtgattag ctatcagcag ctggcggccc tggcgggtaa
     1201 tccggcggcc accgccgccg ttaaaaccgc gctgagcggt aacccggtgc cgattctgat
     1261 tccgtgccat cgtgtggtta gctctagcgg tgcggttggc ggttatgaag gtggtctggc
     1321 ggtgaaagag tggctgctgg cccatgaagg tcatcgtctg ggtaaaccgg gtctgggagg
     1381 tagcggtagc ggtagcggtg gaggtggaag cagcactaga atgagtcgcg atagtgatcc
     1441 catgaaatgg taccacggca acctgtcccg cgaggcagcc gacgaactgc taaagcaggg
     1501 ttacgaggac gggacatttc tggtgcgcga gagcagcaca gcggccgggg actttgtgct
     1561 gagcctgctc tgccagggtg aggtgtgtca ctaccaggtg cggcgacatg gcggcgagga
     1621 cgccttcttc tccatcgacg acaaggtaca gacgaagatc ctgcacggcc tggacacgct
     1681 cgtcgactac tatcagcagg cggccaacgg tctgcccacc aagctgacgg tgcccttgat
     1741 ccgcgatctg ccgccgcaca acactcgcag ccacggagtg accaatctgc tgcatcgcgc
     1801 cacaagtaaa aacgagagca aggtcgtctt cgagctgctc aagtgcggct atcggaattt
     1861 cgatgccaag aatcaggatg gacagacggc tctgcatctg gctgcactcc actcggacga
     1921 ggatatactg aaacacctgt tgaatgcaaa ggtccaggtc aacagctccg actcctttgg
     1981 ctgccagcca ttgcactacg ccgctcgctc caaaccggcc agtttcatac gcacactcat
     2041 ctcggcacag gccaatgtcc agggcaggaa catcgacaac ggctatgtgc cgctgcatga
     2101 ggctgccaag cacggcaacc tggaggctgt gcaagagctg ctcctggctg aggctccgcc
     2161 gttgcccaga acaagttcgg gtgaatttcc attcgatctg gccaaggaag cgggccaaac
     2221 ggcagtagaa gaattcctgc tcaattacaa gctgccaccg gcaaatacca caagagacca
     2281 gtggtatcat ggcactctga ccagagaaga ggcagtagcc atactaaaaa agcatgccaa
     2341 ggagctattg gccaagcaac ctgaagtaga cacctccggc tgctttctgg ttcgctactc
     2401 cgagtcgccg gcggcctcgg gattggttct aacattgctt agtgatcagg tggtcaagaa
     2461 ttttcgtatt tcgcaagctg atctctacca gaatggtaac aaggtgcagt cgggcgggtc
     2521 caagtttctg tacattgatg acggtcccta ctggccaagc gttgaacatc taatagccca
     2581 cttcatgcgt ttctcgtacg ggttgccagt gagtctcaag tatccggttc cgccacagcc
     2641 caagccagag gtgccatcgt ttgccacaat acctcgatct aatatgaagc caaaagcagc
     2701 atcgtaagaa ttcccgggtc gactcgagcg gccgcatcgt gactgactga cgatctgcct
     2761 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2821 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2881 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     2941 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     3001 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3061 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3121 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3181 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3241 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3301 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3361 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3421 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3481 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3541 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3601 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3661 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3721 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3781 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3841 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3901 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3961 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4021 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4081 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4141 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4201 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4261 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4321 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4381 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4441 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4501 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4561 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4621 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4681 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4741 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4801 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4861 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4921 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4981 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     5041 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     5101 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     5161 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     5221 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     5281 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     5341 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     5401 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     5461 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     5521 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     5581 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     5641 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     5701 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     5761 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     5821 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     5881 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     5941 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     6001 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     6061 aagaaaaacc acgctagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6121 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6181 ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6241 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     6301 attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     6361 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     6421 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg
     6481 tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat
     6541 actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac
     6601 gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt
     6661 tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt
     6721 tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc tggcgtcagg
     6781 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     6841 ctcaaggcgc actccc
//
