LOCUS       sequence-258867-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6XHis::dCrk (based on crk isoform RA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  AUTHORS   Spracklen AJ, Thornton-Kolbe EM, Bonner AN, Florea A, Compton PJ, 
            Fernandez-Gonzalez R, Peifer M
  TITLE     The Crk adapter protein is essential for Drosophila embryogenesis, 
            where it regulates multiple actin-dependent morphogenic events.
  JOURNAL   Mol Biol Cell. 2019 Jul 18:mbcE19050302. doi: 
            10.1091/mbc.E19-05-0302.
  PUBMED    31318326
REFERENCE   2  (bases 1 to 6139)
  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: "Mol Biol 
            Cell. 2019 Jul 18:mbcE19050302. doi: 10.1091/mbc.E19-05-0302."
COMMENT     Sequence Label: pET28P_dCrk
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(35..1117)
                     /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     1079..1098
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1118..1195)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1401..1420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1504..1523
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1504..1522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1523..1547
                     /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             1562..1584
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1597..1614
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1618..1641
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2507..2524
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2577..2595)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2591..2638
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2675..3130
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2762..2781)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2972..2993
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3222..4037)
                     /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(3222..4037)
                     /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     3945..3964
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4029..4048
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4159..4747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4648..4667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4901..4918
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4933..5072
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4998..5017)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5158..5180
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5174..5365)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
       61 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      121 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
      181 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
      241 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
      301 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
      361 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
      421 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
      481 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
      541 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
      601 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
      661 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
      721 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
      781 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      841 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      901 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      961 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     1021 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     1081 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     1141 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     1201 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     1261 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     1321 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     1381 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     1441 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     1501 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     1561 ttttgtttaa ctttaagaag gagatatacc atgggtcatc atcatcacca tcatggcctg
     1621 gaagtgctgt ttcagggtcc gggtgggcat atggatacat ttgacgtttc tgataggaac
     1681 agctggtact ttggtcccat gtctagacag gatgctactg aagttttgat gaacgaacgc
     1741 gagcggggag tgtttttagt ccgtgatagt aactcgatag caggggatta tgtactttgt
     1801 gtaagagaag atacaaaagt tagcaactac atcattaaca aagttcaaca acaggatcaa
     1861 atcgtttacc gcattgggga tcagtctttt gacaatctac cgaaactctt aactttttac
     1921 actcttcatt atttggatac aaccccttta aaacggcctg cgtgtagaag ggtggaaaaa
     1981 gtaataggaa agttcgattt cgttggcagc gatcaagatg atttaccttt tcaaagaggt
     2041 gaagttttaa caatagttcg aaaagacgag gatcaatggt ggactgcgcg taactcctcg
     2101 gggaaaattg gtcaaatacc ggttccctat atacaacagt atgacgatta tatggatgaa
     2161 gatgctattg ataaaaacga accttccatt tcgggatcta gcaatgtatt tgaaagtact
     2221 cttaaaagga cagatttaaa tcgaaaacta cctgcatacg cccgcgtaaa acagtcaagg
     2281 gtccctaacg catacgataa gactgcatta aaattggaaa taggtgacat tattaaagtc
     2341 actaaaacaa acattaatgg gcaatgggag ggagaattaa atggaaaaaa tggtcatttt
     2401 cccttcacgc acgttgaatt tgtcgatgat tgtgatttaa gcaaaaactc cacagaaata
     2461 tgctaagaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     2521 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2581 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2641 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2701 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2761 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2821 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2881 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2941 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3001 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3061 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3121 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3181 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     3241 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     3301 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     3361 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     3421 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     3481 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     3541 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     3601 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     3661 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     3721 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     3781 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3841 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3901 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3961 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     4021 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     4081 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4141 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4201 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4261 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4321 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4381 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4441 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4501 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4561 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4621 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4681 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4741 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4801 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4861 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4921 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4981 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     5041 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     5101 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5161 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     5221 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     5281 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     5341 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     5401 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     5461 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     5521 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     5581 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5641 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5701 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5761 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5821 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5881 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5941 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     6001 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     6061 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     6121 gtcgagatcc cggtgccta
//
