LOCUS       sequence-194533-        6124 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 6124)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 6124)
  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: 5KO9 (HMCES)
FEATURES             Location/Qualifiers
     source          1..6124
                     /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)."
     CDS             901..921
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             989..1006
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1059..1077)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1073..1120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1157..1612
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1244..1263)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1454..1475
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1704..2519)
                     /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(1704..2519)
                     /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     2427..2446
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2511..2530
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2641..3229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3130..3149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3383..3400
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3415..3554
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3480..3499)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3640..3662
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3656..3847)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4656..5738)
                     /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     5700..5719
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5739..5816)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6022..6041
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactatg tgtgggcgaa catcctgtca cttacctaga
      121 gatgttctca cgagagcttg cgcctaccag gatcggcggg gccagcagcg gctcccggag
      181 tggagggacc ctgataagta ctgcccctct tacaacaaga gtcctcaatc caacagccca
      241 gtgcttctgt ctcgactgca ctttgagaag gatgcagact catctgagcg tatcattgct
      301 cccatgcgct ggggcttggt cccttcttgg ttcaaagaaa gtgatccttc caagctgcag
      361 ttcaatacta ccaactgtcg tagtgatacc gtaatggaga aacggtcatt taaggtgcct
      421 ctgggaaagg gaagacgctg tgtcgtttta gcagatggat tctatgagtg gcagcgatgt
      481 cagggaacaa accagaggca gccatacttc atctattttc ctcaaatcaa gacagagaag
      541 tcaggtagca ttggtgctgc agatagtcct gagaactggg agaaagtctg ggacaactgg
      601 aggctgctga caatggccgg gatctttgac tgctgggagc ccccagaggg aggagatgtc
      661 ctgtattcct ataccatcat cacagtggat tcctgcaaag gcttgagtga catccaccac
      721 aggatgcctg ccatattaga tggagaggag gcagtttcta aatggcttga ctttggtgaa
      781 gtctcaactc aggaagctct gaaattaatc cacccaacag agaacatcac cttccatgca
      841 gtctcttctg tggtgaacaa ctcgcgaaac aacactcctg agtgtctggc tcctgtcgca
      901 gagaacctct acttccaatc gcaccatcat caccaccatt gaggatccga attcgagctc
      961 cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
     1021 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1081 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1141 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1201 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1261 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1321 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1381 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1441 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1501 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1561 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     1621 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1681 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1741 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1801 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1861 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1921 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1981 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2041 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2101 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2161 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     2221 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2281 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2341 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2401 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2461 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2521 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     2581 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2641 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2701 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2761 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2821 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2881 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2941 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3001 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3061 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3121 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3181 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3241 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3301 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3361 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3421 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3481 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3541 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3601 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3661 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3721 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3781 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3841 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3901 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3961 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4021 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4081 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4141 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4201 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4261 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4321 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4381 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4441 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4501 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4561 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4621 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4681 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4741 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4801 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4861 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4921 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4981 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5041 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5101 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5161 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5221 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5281 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5341 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5401 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5461 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5521 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5581 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5641 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5701 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5761 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5821 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5881 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5941 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6001 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6061 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6121 aaat
//
