LOCUS       sequence-296542-        6139 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression/crystallography.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  TITLE     Opher Gileadi SGC expression vectors
REFERENCE   2  (bases 1 to 6139)
  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: TBXTA-c022
FEATURES             Location/Qualifiers
     source          1..6139
                     /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             959..1003
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1004..1021
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1074..1092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1088..1135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1172..1627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1259..1278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1469..1490
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1719..2534)
                     /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(1719..2534)
                     /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     2442..2461
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2526..2545
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2656..3244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3145..3164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3398..3415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3430..3569
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3495..3514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3655..3677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3671..3862)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4671..5753)
                     /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     5715..5734
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5754..5831)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6037..6056
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gtgcagcagc catcatcatc atcatcacgg
      121 cagcggcagc ggctctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa
      181 ggaaggtgaa tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa
      241 agtgaaaatg acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt
      301 tccaatgaat tcactcaggt ttctctttga gggtcagaga attgctgata atcatactcc
      361 aaaagaactg ggaatggagg aagaagatgt gattgaagtt taccaggagc aaacgggagg
      421 tggagaactg cgtgtgggtc tggaagaaag cgaactgtgg ctgcgcttta aagagctgac
      481 caacgaaatg atcgtgacca aaaacggccg ccgcatgttt cctgtgctga aagttaacgt
      541 gagcggcctg gacccgaacg ccatgtacag cttcctgctg gatttcgttg ccgcagataa
      601 ccaccgctgg aagtatgtga acggcgaatg ggtgccgggt ggcaaaccgg aaccgcaggc
      661 acctagctgc gtgtatatcc acccggatag ccctaacttc ggtgcccatt ggatgaaagc
      721 cccggtgagc ttcagcaagg tgaagctgac caacaaactg aacggcggcg gccaaatcat
      781 gctgaacagc ttacataaat atgaacctcg catccacatt gtgcgtgtgg gcgacccgca
      841 acgtatgatc accagccatt gcttcccgga gacacagttc attgccgtta ccgcctacca
      901 gaacgaggaa attaccgcac tgaaaattaa atacaaccct tttgcatgaa gcggtggcgg
      961 tctgaacgac atcttcgagg ctcagaaaat cgaatggcac gaacaccacc accaccacca
     1021 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1081 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1141 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1201 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1261 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1321 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1381 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1441 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1501 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1561 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1621 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1681 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1741 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1801 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1861 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1921 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1981 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2041 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2101 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2161 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2221 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2281 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2341 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2401 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2461 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2521 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     2581 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2761 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3301 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3361 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3421 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3481 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3541 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3601 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3661 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3721 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3781 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3841 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3901 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3961 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4021 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4081 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4141 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4201 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4261 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4321 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4381 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4441 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4501 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4561 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4621 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4681 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4741 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4801 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4861 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4921 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4981 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5041 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5101 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5161 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5221 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5281 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5341 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5401 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5461 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5521 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5581 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5641 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5701 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5761 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5821 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5881 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5941 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     6001 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6061 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     6121 gatctcgatc ccgcgaaat
//
