LOCUS       pET28_HisSUMO_Lc        5989 bp DNA     circular SYN 28-DEC-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5989)
  AUTHORS   Columbia University
  TITLE     Direct Submission
  JOURNAL   Exported Dec 28, 2023 from SnapGene Viewer 7.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5989
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     rep_origin      13..468
                     /direction=RIGHT
                     /locus_tag="f1 origin"
                     /label=f1 origin
                     /note="f1 origin"
                     /note="color: #999999"
                     /note="color: #999999"
                     /note="color: #c0c0c0"
     CDS             complement(561..1376)
                     /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"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(564..1376)
                     /locus_tag="Kan"
                     /label=Kan
                     /note="Kan"
                     /note="color: #e9d024; direction: LEFT"
                     /note="color: #e9d024; direction: LEFT"
                     /note="color: #0000ff; direction: LEFT"
     rep_origin      1475..2118
                     /direction=RIGHT
                     /locus_tag="pBR322 origin"
                     /label=pBR322 origin
                     /note="pBR322 origin"
                     /note="color: #999999"
                     /note="color: #999999"
                     /note="color: #c0c0c0"
     rep_origin      1498..2086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    2272..2414
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(2516..2707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3516..4598)
                     /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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     CDS             complement(3519..4598)
                     /locus_tag="LacI"
                     /label=LacI
                     /note="LacI"
                     /note="color: #e9d024; direction: LEFT"
                     /note="color: #e9d024; direction: LEFT"
                     /note="color: #0000ff; direction: LEFT"
     promoter        complement(4599..4676)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
     promoter        4989..5032
                     /locus_tag="T7Lac"
                     /label=T7Lac
                     /note="T7Lac"
                     /note="color: #346ee0"
                     /note="color: #346ee0"
                     /note="color: #8080ff"
     promoter        4989..5007
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        4989..5005
                     /locus_tag="T7 promoter"
                     /label=T7 promoter
                     /note="T7 promoter"
                     /note="color: #346ee0"
                     /note="color: #346ee0"
                     /note="color: #8080ff"
     misc_binding    5008..5032
                     /locus_tag="Lac Operator"
                     /label=Lac Operator
                     /note="Lac Operator"
                     /note="color: #ff4809"
                     /note="color: #ff4809"
                     /note="color: #f7a68a"
     protein_bind    5008..5032
                     /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)."
                     /note="color: #31849b"
     RBS             5047..5069
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     CDS             5077..5433
                     /codon_start=1
                     /locus_tag="6HSumo"
                     /label=6HSumo
                     /note="6HSumo"
                     /note="color: #e9d024"
                     /note="color: #e9d024"
                     /note="color: #0000ff"
                     /translation="MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHI
                     NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDM
                     EDNDIIEAHREQIGG"
     CDS             5089..5106
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     CDS             5116..5133
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /note="color: #cc99b2
                     Cleavage site after base 5127"
                     /translation="LVPRGS"
     CDS             5140..5433
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="color: #cc99b2
                     Cleavage site after base 5433"
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     5413..5449
                     /label=DG_Lck_SH2_GA_for
                     /note="color: black; sequence: 
                     CACAGAGAACAGATTGGTGGTGCGAACAGCCTGGAGC; added: 2022-02-02"
     primer_bind     complement(5413..5433)
                     /label=His_sumo_BB_rv
                     /note="color: black; sequence: ACCACCAATCTGTTCTCTGTG; 
                     added: 2022-02-02"
     CDS             5446..5748
                     /codon_start=1
                     /label=SH2 Lck
                     /note="color: #993366"
                     /translation="EPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSV
                     RDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRP"
     primer_bind     complement(5744..5781)
                     /label=DG_Lck_SH2_GA_rev
                     /note="color: black; sequence: 
                     CGCTTCAAAAATATCGTTCAGGCCGGTCTGGCAGGGGC; added: 2022-02-02"
     primer_bind     complement(5744..5757)
                     /label=SH2 boundry rev
                     /note="color: black; sequence: GGTCTGGCAGGGGC; added: 
                     2022-01-25"
     primer_bind     5758..5823
                     /label=his_sumo_BB_fw
                     /note="color: black; sequence: 
                     GGCCTGAACGATATTTTTGAAGCGCAGAAAATTGAATGGCATGAATAACGTCGACAAGC
                     TTGCGGC; added: 2022-01-24"
     primer_bind     5758..5823
                     /label=Downstr_AVI_GA intro for primer
                     /note="color: black; sequence: 
                     GGCCTGAACGATATTTTTGAAGCGCAGAAAATTGAATGGCATGAATAACGTCGACAAGC
                     TTGCGGC; added: 2022-02-02"
     CDS             5758..5802
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /note="color: #cc99b2"
                     /translation="GLNDIFEAQKIEWHE"
     misc_feature    5803..5805
                     /label=Grb2 SH2-C-SH3
                     /note="color: #a6acb3; direction: RIGHT"
     CDS             5834..5851
                     /codon_start=1
                     /locus_tag="His Tag"
                     /label=His Tag
                     /note="His Tag"
                     /note="color: #e9d024"
                     /note="color: #e9d024"
                     /note="color: #ff8000"
                     /translation="HHHHHH"
     terminator      5918..5965
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     terminator      5919..5965
                     /locus_tag="T7 term"
                     /label=T7 term
                     /note="T7 term"
                     /note="color: #9d1b1c"
                     /note="color: #9d1b1c"
                     /note="color: #000000"
ORIGIN
        1 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
       61 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      121 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      181 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      241 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      301 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      361 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      421 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
      481 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      541 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      601 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      661 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      721 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      781 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
      841 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
      901 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
      961 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1021 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     1081 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1141 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     1201 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     1261 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     1321 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     1381 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     1441 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1501 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1561 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1621 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1681 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1741 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1801 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1861 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     1921 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     1981 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2041 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2101 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2161 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2221 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2281 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     2341 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2401 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2461 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2521 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     2581 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     2641 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     2701 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2761 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2821 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2881 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     2941 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3001 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3061 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3121 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3181 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3241 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3301 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3361 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3421 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3481 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3541 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3601 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3661 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3721 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     3781 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     3841 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     3901 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     3961 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4021 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4081 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4141 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4201 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4261 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4321 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4381 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4441 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4501 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4561 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4621 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4681 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4741 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     4801 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     4861 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     4921 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     4981 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     5041 aataattttg tttaacttta agaaggagat atacatatgg gcagcagcca tcatcatcat
     5101 catcacggca gcggcctggt gccgcgcggc agcgctagca tgtcggactc agaagtcaat
     5161 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
     5221 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
     5281 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
     5341 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
     5401 attattgagg ctcacagaga acagattggt ggtgcgaaca gcctggagcc cgaaccctgg
     5461 ttcttcaaga acctgagccg caaggacgcg gagcggcagc tcctggcgcc cgggaacact
     5521 cacggctcct tcctcatccg ggagagcgag agcaccgcgg gatcgttttc actgtcggtc
     5581 cgggacttcg accagaacca gggagaggtg gtgaaacatt acaagatccg taatctggac
     5641 aacggtggct tctacatctc ccctcgaatc acttttcccg gcctgcatga actggtccgc
     5701 cattacacca atgcttcaga tgggctgtgc acacggttga gccgcccctg ccagaccggc
     5761 ctgaacgata tttttgaagc gcagaaaatt gaatggcatg aataacgtcg acaagcttgc
     5821 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     5881 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     5941 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccgga
//
