LOCUS       sequence-193505-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  TITLE     SGC Toronto Q4 2016, Q1 Q2 2017
REFERENCE   2  (bases 1 to 6422)
  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: 2QO2 (EPHA3)
FEATURES             Location/Qualifiers
     source          1..6422
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1287..1304
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1357..1375)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1371..1418
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1455..1910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1542..1561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1752..1773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2002..2817)
                     /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(2002..2817)
                     /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     2725..2744
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2809..2828
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2939..3527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3428..3447
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3681..3698
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3713..3852
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3778..3797)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3938..3960
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3954..4145)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4954..6036)
                     /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     5998..6017
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6037..6114)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6320..6339
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg ttccgcgtgg tagtgatgaa aaaagacttc attttggcaa tgggcattta
      181 aaacttccag gtctcaggac ttatgttgac ccacatacat atgaagaccc tacccaaact
      241 gttcatgagt ttgccaagga attggatgcc accaacatat ccattgataa agttgttgga
      301 gcaggtgaat ttggagaggt gtgcagtggt cgcttaaaac ttccttcaaa aaaagagatt
      361 tcagtggcca ttaagaccct gaaagttggc tacacagaaa agcagaggag agacttcctg
      421 ggagaagcaa gcattatggg acagtttgac caccccaata tcattcgact ggaaggagtt
      481 gttaccaaaa gtaagccagt tatgattgtc acagaataca tggagaatgg ttccttggat
      541 agtttcctac gtaaacacga tgcccagttt actgtcattc agctagtggg gatgcttcga
      601 gggatagcat ctggcatgaa gtacctgtca gacatgggct atgttcaccg agacctcgct
      661 gctcggaaca tcttgatcaa cagtaacttg gtgtgtaagg tttctgattt cggactttcg
      721 cgtgtcctgg aggatgaccc agaagctgct tatacaacaa gaggagggaa gatcccaatc
      781 aggtggacat caccagaagc tatagcctac cgcaagttca cgtcagccag cgatgtatgg
      841 agttatggga ttgttctctg ggaggtgatg tcttatggag agagaccata ctgggagatg
      901 tccaatcagg atgtaattaa agctgtagat gagggctatc gactgccacc ccccatggac
      961 tgcccagctg ccttgtatca gctgatgctg gactgctggc agaaagacag gaacaacaga
     1021 cccaagtttg agcagattgt tagtattctg gacaagctta tccggaatcc cggcagcctg
     1081 aagatcatca ccagtgcagc cgcaaggcca tcaaaccttc ttctggacca aagcaatgtg
     1141 gatatcacta ccttccgcac aacaggtgac tggcttaatg gtgtctggac agcacactgc
     1201 aaggaaatct tcacgggtgt ggagtacagt tcttgtgaca caatagccaa gatttcctga
     1261 tgacgaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1321 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1381 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1441 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1501 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1561 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1621 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1681 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1741 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1801 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1861 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1921 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1981 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2041 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2101 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2161 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2221 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2281 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2341 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2401 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2461 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2521 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2581 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2641 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2701 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2761 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     2821 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     2881 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2941 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3001 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3061 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3121 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3181 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3241 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3301 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3361 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3421 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3481 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3541 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3601 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3661 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3721 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3781 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3841 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3901 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3961 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4021 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4081 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4141 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4201 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4261 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4321 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4381 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4441 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4501 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4561 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4621 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4681 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4741 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4801 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4861 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4921 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4981 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5041 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5101 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5161 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5221 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5281 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5341 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5401 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5461 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5521 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5581 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5641 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5701 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5761 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5821 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5881 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5941 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6001 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6061 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6121 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6181 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6241 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6301 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6361 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     6421 at
//
