LOCUS       sequence-296582-        5963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression/Streptavidin pull-down.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5963)
  TITLE     Opher Gileadi SGC expression vectors
REFERENCE   2  (bases 1 to 5963)
  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"
COMMENT     Sequence Label: TBXTA-c044
FEATURES             Location/Qualifiers
     source          1..5963
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             731..775
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             828..845
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(898..916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      912..959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      996..1451
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1083..1102)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1293..1314
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1543..2358)
                     /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(1543..2358)
                     /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     2266..2285
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2350..2369
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2480..3068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2969..2988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3222..3239
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3254..3393
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3319..3338)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3479..3501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3495..3686)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4495..5577)
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5539..5558
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5578..5655)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5861..5880
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggaa ctgcgtgtgg gtctggaaga
      181 aagcgaactg tggctgcgct ttaaagagct gaccaacgaa atgatcgtga ccaaaaacgg
      241 ccgccgcatg tttcctgtgc tgaaagttaa cgtgagcggc ctggacccga acgccatgta
      301 cagcttcctg ctggatttcg ttgccgcaga taaccaccgc tggaagtatg tgaacggcga
      361 atgggtgccg ggtggcaaac cggaaccgca ggcacctagc tgcgtgtata tccacccgga
      421 tagccctaac ttcggtgccc attggatgaa agccccggtg agcttcagca aggtgaagct
      481 gaccaacaaa ctgaacggcg gcggccaaat catgctgaac agcttacata aatatgaacc
      541 tcgcatccac attgtgcgtg tgggcgaccc gcaacgtatg atcaccagcc attgcttccc
      601 ggagacacag ttcattgccg ttaccgccta ccagaacgag gaaattaccg cactgaaaat
      661 taaatacaac ccttttgcaa aagccttctt agatgccaaa gagcgcagcg acagcagtaa
      721 aggtggatac ggcctgaatg atatctttga agcgcagaag attgaatggc atgaatgata
      781 aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
      841 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      901 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      961 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     1021 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1081 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1141 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1201 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1261 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     1321 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1381 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1441 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1501 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     1561 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1621 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1681 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1741 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1801 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     1861 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     1921 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     1981 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     2041 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     2101 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2161 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2221 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     2281 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     2341 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     2401 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2461 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2521 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2581 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2641 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2701 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2761 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2821 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2881 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2941 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3001 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3061 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3121 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3181 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3241 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3301 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     3361 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3421 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3481 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3541 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3601 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3661 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3721 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3781 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3841 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3901 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3961 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4021 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4081 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4141 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4201 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     4261 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     4321 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     4381 atagtcatgt cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     4441 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4561 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4621 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     4681 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     4741 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     4801 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     4861 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     4921 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4981 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     5041 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     5101 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5161 ggcagcttcc acagcaatgg catcctggtc atccagctga tagttaatga tcagcccact
     5221 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     5281 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     5341 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     5401 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     5461 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     5521 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     5581 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     5641 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     5701 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     5761 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     5821 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5881 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5941 tcgagatctc gatcccgcga aat
//
