LOCUS       sequence-346799-        6077 bp    DNA     circular SYN 15-APR-2025
DEFINITION  express murine Syk tandem SH2 domains (Ser 8 to Gln 264), with R44A 
            mutation and a TEV-cleavable His-tag. For use in E.coli strain 
            Rosetta 2 (DE3)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6077)
  TITLE     mutants for salt bridges found in syk md simulations
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6077)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Syk tSH2 R44A in pET-30a(+)
FEATURES             Location/Qualifiers
     source          1..6077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..160
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             938..955
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1008..1026)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1022..1069
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1106..1561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1193..1212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1403..1424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1653..2468)
                     /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(1653..2468)
                     /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     2376..2395
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2460..2479
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2590..3178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3079..3098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3332..3349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3364..3503
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3429..3448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3589..3611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3605..3796)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4605..5687)
                     /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     5649..5668
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5688..5765)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5971..5990
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggtagtagt catcatcatc atcaccatga
      121 ttatgatatt ccgaccaccg aaaatctgta ttttcagagt gcaaatcatc tgacctattt
      181 ctttggcaat attacccgtg aagaagccga agattatctg gttcagggtg gcatgaccga
      241 tggtctgtat ctgctgcgtc agagtgcaaa ttatctgggt ggttttgcac tgagcgttgc
      301 acataatcgt aaagcccatc attataccat tgaacgcgaa ctgaatggta cctatgcaat
      361 tagtggtggt cgcgcccatg ccagtccggc agatctgtgt cattatcata gtcaggaacc
      421 ggatggcctg atttgtctgc tgaaaaaacc gtttaatcgt ccgccgggcg ttcagccgaa
      481 aaccggccct tttgaagatc tgaaagaaaa tctgattcgc gaatatgtta aacagacctg
      541 gaatctgcag ggtcaggccc tggaacaggc aattattagt cagaaaccgc agctggaaaa
      601 actgattgca accaccgcac atgaaaaaat gccgtggttt catggcaata ttagtcgtga
      661 tgaaagcgaa cagaccgttc tgattggcag taaaaccaat ggtaaatttc tgattcgcgc
      721 ccgtgataat agcggtagct atgccctgtg cctgctgcat gaaggtaaag ttctgcatta
      781 tcgtattgat cgtgataaaa ccggtaaact gagtattccg gaaggcaaaa aatttgatac
      841 cctgtggcag ctggttgaac attatagcta taaaccggat ggtctgctgc gcgtgctgac
      901 cgttccgtgc cagaaaattg gcgcccagta actcgagcac caccaccacc accactgaga
      961 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     1021 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     1081 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1141 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1201 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1261 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1321 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1381 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1441 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1501 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1561 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1621 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     1681 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     1741 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     1801 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     1861 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     1921 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     1981 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     2041 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     2101 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     2161 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     2221 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     2281 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     2341 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2401 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     2461 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     2521 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2581 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2641 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2701 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     2761 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2821 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2881 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2941 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3001 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3061 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3121 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3181 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3241 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3301 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3361 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3421 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3481 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     3541 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3601 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     3661 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     3721 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     3781 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     3841 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3901 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     3961 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4021 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4081 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4141 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4201 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4261 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4321 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4381 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4441 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4501 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4561 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4741 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4801 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4861 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     4921 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4981 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5041 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5101 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5161 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5221 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5281 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5341 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5401 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5461 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5521 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5581 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5641 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5701 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5761 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5821 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5881 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5941 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     6001 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     6061 tctcgatccc gcgaaat
//
