LOCUS       sequence-458419-        6657 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of  human LYN Kinase in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6657)
  TITLE     Mesecar lab plasmids 2024
REFERENCE   2  (bases 1 to 6657)
  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"
COMMENT     Sequence Label: pET28a_LYN
FEATURES             Location/Qualifiers
     source          1..6657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..31
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             41..61
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1436..1453
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1506..1524)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1520..1567
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1604..2059
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1691..1710)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1901..1922
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2151..2966)
                     /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(2151..2966)
                     /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     2874..2893
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2958..2977
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3088..3676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3577..3596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3830..3847
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3862..4001
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3927..3946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4087..4109
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4103..4294)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5103..6185)
                     /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     6147..6166
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6186..6263)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6469..6488
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6572..6591
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6572..6590
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6591..6615
                     /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             6630..6652
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catgggcagc agccatcatc atcatcatca cagcagcggc gagaatcttt attttcaggg
       61 ccatatggaa caaggagata tagtagttgc tctatatcca tacgacggca ttcatccgga
      121 tgacctgtcg ttcaagaagg gtgagaagat gaaagttctg gaggagcacg gcgaatggtg
      181 gaaagccaaa agcttgttaa ccaaaaaaga aggttttatc ccgagcaatt atgtcgcgaa
      241 gctgaacacc ctggagacgg aagagtggtt cttcaaggac atcacccgta aagacgctga
      301 acgccaactg ctggcaccgg gtaactccgc gggtgcgttt ctgattcgcg aaagcgaaac
      361 tctgaaaggc agcttcagct tgtccgtgcg tgattttgat ccggttcatg gtgatgtgat
      421 caagcactat aaaatccgca gcctcgacaa cggcggttat tacatctccc cgcgtattac
      481 ctttccgtgc atctctgaca tgattaagca ctaccagaag caagctgatg gcttgtgccg
      541 tcgtctggag aaggcgtgta tttctccgaa accgcagaag ccgtgggata aagaccatat
      601 gctcgaggct tgggaaatac ccagggagtc aattaaactg gttaagcgtc tgggtgctgg
      661 tcaatttggc gaggtgtgga tgggttatta taacaactcg accaaagtgg ccgtgaagac
      721 gttaaagccg ggcaccatga gcgtccaggc gtttttggaa gaggcgaatc tgatgaagac
      781 gttgcagcat gacaaattag tgcgcttgta cgcggttgtt actcgcgagg agccgatcta
      841 cattatcacc gaatatatgg caaagggcag cctgctggac tttctgaaga gcgatgaggg
      901 tggtaaagtg ctcctgccta aactgatcga cttctctgcc cagattgcgg aaggcatggc
      961 gtatattgaa cgtaaaaact atatccaccg cgatctgcgt gcagcaaacg ttttggttag
     1021 cgagtccctg atgtgcaaaa tcgcggattt cggtttagcg cgcgtgattg aggacaacga
     1081 gtacaccgcg agagagggcg ctaagtttcc gattaaatgg accgcgccag aagcgatcaa
     1141 cttcggctgc ttcaccatca agtccgacgt ttggtctttc ggcattcttt tgtacgagat
     1201 cgtcacctac ggtaaaatcc cgtatccggg ccgtaccaat gcagacgtga tgacggctct
     1261 gagccagggt taccgtatgc cgcgtgttga aaattgtccg gatgaactgt acgatattat
     1321 gaagatgtgc tggaaagaaa aagcggaaga acgtccgacc tttgattacc tgcagagcgt
     1381 tctggacgac ttctatactg ctaccgaggg tcaataccaa caacagccgc tcgagcacca
     1441 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1501 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1561 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1621 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1681 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1741 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1801 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1861 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1921 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1981 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2041 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     2161 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2221 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2281 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2341 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2401 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2461 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2521 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2581 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2641 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2701 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2761 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2821 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2881 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2941 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3001 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3061 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3121 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3181 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3241 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3301 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3361 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3421 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3481 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3541 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3601 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3661 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3721 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3781 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3841 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3901 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3961 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4021 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4081 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4141 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4201 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4261 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4321 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4381 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4441 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4501 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4561 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4621 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4681 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4741 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4801 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4861 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4921 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4981 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5041 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5101 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5161 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5221 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5281 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5341 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5401 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5461 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5521 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5581 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5641 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5701 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5761 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5821 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5881 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5941 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     6001 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     6061 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6121 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6181 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6241 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6301 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6361 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6421 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6481 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6541 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     6601 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatac
//
