LOCUS       sequence-136205-        6004 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 6004)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 6004)
  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: SPIN4
FEATURES             Location/Qualifiers
     source          1..6004
                     /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             865..882
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(935..953)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      949..996
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1033..1488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1120..1139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1330..1351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1581..2396)
                     /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(1581..2396)
                     /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     2304..2323
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2388..2407
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2518..3106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3007..3026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3260..3277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3292..3434
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3360..3379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3520..3542
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3536..3727)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4536..5618)
                     /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     5580..5599
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5619..5696)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5902..5921
                     /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 atccatgact cgtaggaaca tcgtgggctg
      181 ccgcattcaa cacggctgga aggaaggcaa cgagccagtg gagcagtgga agggtactgt
      241 gctcgagcag gtttccgtga agcccactct ttacatcatt aaatatgatg gcaaagatag
      301 tgtgtatgga ctagaactgc accgcgataa gagagtttta gcgctagaga tccttcctga
      361 gagagtgcca actcctcgta tcgattcacg actggcagat tccctgattg gcaaggcagt
      421 ggagcatgtg tttgaaggtg aacatggtac caaggatgaa tggaagggta tggtcctggc
      481 gcgagctcct gtgatggata cttggtttta catcacctac gagaaagatc ctgttctcta
      541 tatgtacacg ctgcttgatg actacaaaga tggtgactta cgcattattc cagattccaa
      601 ctactatttc cctacagcag aacaggagcc tggagaggtg gtcgacagtc tcgtgggcaa
      661 gcaggtggag catgccaaag atgacgggtc caagagaact ggcattttta tacatcaagt
      721 ggtggcgaag ccatctgttt acttcattaa gtttgatgat gatattcaca tttatgtcta
      781 tggtttggtg aaaactcctt gacagtaaag gtggatacgg atccgaattc gagctccgtc
      841 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
      901 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
      961 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1021 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1081 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1141 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1201 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1261 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1321 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1381 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1441 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt
     1501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1561 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1621 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     1681 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     1741 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     1801 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1861 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1921 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     1981 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2041 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2101 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2161 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2221 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2281 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2341 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2401 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2461 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2521 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2581 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2641 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2701 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2761 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2821 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2881 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2941 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3001 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3061 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3121 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3181 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3241 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3301 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     3361 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3421 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3481 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3541 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3601 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3661 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3721 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3781 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3841 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3901 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3961 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4021 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4081 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4141 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4201 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4261 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4321 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4381 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4441 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4501 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4561 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4621 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4681 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4741 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4801 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4861 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4921 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4981 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5041 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5101 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5161 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5221 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5281 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5341 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5401 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5461 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5521 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5581 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5641 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5701 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5761 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5821 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5881 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5941 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6001 aaat
//
