LOCUS       sequence-251763-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His10-Src42a for purification from E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  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 6933)
  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: pET His10-Src42a
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     151..170
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        151..169
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    170..194
                     /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             209..231
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             250..279
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             289..306
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             310..342
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1948..1965
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2018..2036)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2032..2079
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2116..2571
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2203..2222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2413..2434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2663..3478)
                     /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(2663..3478)
                     /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     3386..3405
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3470..3489
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3600..4188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4089..4108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4342..4359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4374..4513
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4439..4458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4599..4621
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4615..4806)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5615..6697)
                     /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     6659..6678
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6698..6775)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
       61 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
      121 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      181 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
      241 ggcagcagcc atcatcatca tcatcatcat caccatcaca gcagcggcct ggtgccgcgc
      301 ggcagccata tggctagcat gactggtgga cagcaaatgg gtcgcggatc cgccatgggt
      361 aactgcctca ccacacagaa gggcgaaccc gacaagcccg cagatcgaat caagctggac
      421 gacccgccca ccatcggagt cggagtgggc gtgccacaaa tccccatgcc ctcacacgcc
      481 ggacagccac cggagcagat acgtccggtt ccccagatcc cggagagcga aacggcaggt
      541 gccaacgcca agatttttgt cgccctctac gactacgacg cccgcaccga cgaggatttg
      601 agcttccgca agggagagca cttggagata ctgaatgaca cgcagggtga ctggtggctg
      661 gcgcggagca agaagacacg ttcggaaggc tacattccat ccaattatgt ggccaagttg
      721 aaatcaatcg aagcagaacc ctggtacttc cgcaaaatca aacgcattga ggctgagaaa
      781 aaacttctac tgccagagaa cgagcacggt gcatttttaa ttcgcgattc cgaaagccgt
      841 cacaacgact actcgctatc agtgcgcgat ggcgatacgg ttaagcatta tcgcatcaga
      901 caattggacg aaggcggctt cttcatcgcc aggcgcacga cattcagaac ccttcaggag
      961 ctggtggaac actattcgaa ggactctgat ggcctatgcg tcaacctctg caagccgtgt
     1021 gtccagatcg agaagcctgt aactgagggg ctttcgcacc gcactcgcga tcagtgggag
     1081 atcgacagaa catccttgaa attcgtgcgc aaactgggct ccggacagtt tggcgatgtc
     1141 tgggagggat tgtggaacaa cacaacacct gtggcaatta aaactctgaa atctggcaca
     1201 atggacccca aggatttctt agcggaagcc cagatcatga agaaactgcg ccacaccaag
     1261 cttatacagt tgtacgctgt ctgcactgtt gaggagccta tctatattat cacagagtta
     1321 atgaagcacg gttcactgtt ggaatatctc caagccattg caggcaaggg tcgtagcctt
     1381 aaaatgcaaa ctctgattga tatggcagcg caaatagctg ctggcatggc ttacttggag
     1441 tcccagaatt atattcatag ggatttagcg gcgcgcaatg tactggtagg cgatggaaac
     1501 atcgtcaaaa tcgccgactt tggtttagct aggctcatca aggaggacga atacgaggcg
     1561 cgggtaggcg ccagatttcc cataaaatgg accgctccag aggctgctaa ctacagcaaa
     1621 ttctcaataa aatcggatgt ttggagcttt ggcattcttc tcacagaact ggtcacctac
     1681 ggacgcatac catatccagg catgaccaac gctgaggtgc taacgcaagt ggagcacggc
     1741 tatcgaatgc cgcaacctcc caactgcgag ccgcgcctgt atgagattat gctggaatgt
     1801 tggcacaagg accccatgcg cagacccacg tttgagacgc tacaatggaa actggaagac
     1861 ttctatacat ctgatcagag cgactacaaa gaggcgcagg cctactagaa ttcgagctcc
     1921 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     1981 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2041 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2101 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2161 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2221 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2281 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2341 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2401 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2461 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2521 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2641 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2701 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2761 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2821 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2881 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2941 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3001 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3061 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3121 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3181 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3241 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3301 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3361 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3421 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3481 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3541 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3721 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4261 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4321 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4381 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4441 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4501 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4561 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4621 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4681 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4741 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4801 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4861 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4921 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4981 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5041 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5101 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5161 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5221 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5281 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5341 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5401 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5461 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5521 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5581 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5641 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5701 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5761 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5821 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5881 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5941 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6001 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6061 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6121 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6181 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6241 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6301 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6361 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6421 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6481 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6541 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6601 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6661 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6721 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6781 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6841 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6901 ccggccacgg ggcctgccac catacccacg ccg
//
