LOCUS       sequence-193418-        5661 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 5661)
  TITLE     SGC Oxford 2017
REFERENCE   2  (bases 1 to 5661)
  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: IPCEF1 (PIP3EA-c002)
FEATURES             Location/Qualifiers
     source          1..5661
                     /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             526..543
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(596..614)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      610..657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      694..1149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(781..800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     991..1012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1241..2056)
                     /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(1241..2056)
                     /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     1964..1983
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2048..2067
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2178..2766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2667..2686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2920..2937
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2952..3091
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3017..3036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3177..3199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3193..3384)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4193..5275)
                     /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     5237..5256
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5276..5353)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5559..5578
                     /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 atccatggct gactgccaag ggtggctgta
      181 taagaaaaag gaaaagggaa gtttcctaag caacaaatgg aaaaagttct gggtgatact
      241 gaaggggtcg tcactgtact ggtatagcaa tcaaatggca gagaaagctg atggatttgt
      301 caacctgcct gatttcactg tggaaagagc atctgaatgc aagaaaaagc atgcttttaa
      361 gatcagccat ccacagatca agacctttta ttttgcagct gagaatgtgc aggaaatgaa
      421 cgtgtggtta aataaacttg gatcggctgt aatccatcag tgacagtaaa ggtggatacg
      481 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
      541 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      601 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      661 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      721 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      781 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      841 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      901 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      961 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1021 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1081 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1141 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1201 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1261 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1321 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1381 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1441 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1501 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1561 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1621 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1681 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1741 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1801 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1861 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1921 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1981 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2041 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2101 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2161 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2221 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2281 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2341 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2401 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2461 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2521 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2581 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2641 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2701 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2761 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2821 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2881 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2941 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3001 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3061 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3121 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3181 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3241 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3301 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3361 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3421 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3481 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3541 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3601 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3661 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     3721 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     3781 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     3841 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     3901 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     3961 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4021 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4081 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4141 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4201 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4261 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4321 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4381 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4441 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4501 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4561 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4621 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4681 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4741 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4801 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4861 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4921 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4981 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5041 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5101 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5161 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5221 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5281 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5341 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5401 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5461 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5521 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5581 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5641 gagatctcga tcccgcgaaa t
//
