LOCUS       sequence-398289-        6471 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 6471)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 6471)
  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: PIP4K2AA
FEATURES             Location/Qualifiers
     source          1..6471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     227..246
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        227..245
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    246..270
                     /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             285..307
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             318..335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1562..1579
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1632..1650)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1646..1693
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1730..2185
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1817..1836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2027..2048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2277..3092)
                     /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(2277..3092)
                     /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     3000..3019
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3084..3103
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3214..3802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3703..3722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3956..3973
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3988..4127
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4053..4072)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4213..4235
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4229..4420)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5229..6311)
                     /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     6273..6292
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6312..6389)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
       61 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
      121 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      181 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
      241 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
      301 aaggagatat acatatgcac catcatcatc atcattcttc tggtgtagat ctgggtaccg
      361 agaacctgta cttccaatcc atggacccgc tgctcagcgt cctcatgtgg ggggtaaacc
      421 actcgatcaa tgaactgagc catgttcaaa tccctgttat gttgatgcca gatgacttca
      481 aagcctattc aaaaataaag gtggacaatc acctttttaa caaagaaaac atgccgagcc
      541 atttcaagtt taaggaatac tgcccgatgg tcttccgtaa cctgcgggag aggtttggaa
      601 ttgatgatca agatttccag aattccctga ccaggagcgc acccctcccc aacgactccc
      661 aggcccgcag tggagctcgt tttcacactt cctacgacaa aagatacatc atcaagacta
      721 ttaccagtga agacgtggcc gaaatgcaca acatcctgaa gaaataccac cagtacatag
      781 tggaatgtca tgggatcacc cttcttcccc agttcttggg catgtaccgg cttaatgttg
      841 atggagttga aatatatgtg atagttacaa gaaatgtatt cagccaccgt ttgtctgtgt
      901 ataggaaata cgacttaaag ggctctacag tggctagaga agctagtgac aaagaaaagg
      961 ccaaagaact gccaactctg aaagataatg atttcattaa tgagggccaa aagatttata
     1021 ttgatgacaa caacaagaag gtcttcctgg aaaaactaaa aaaggatgtt gagtttctgg
     1081 cccagctgaa gctcatggac tacagtctgc tggtgggaat tcatgatgtg gagagagccg
     1141 aacaggagga agtggagtgt gaggagaacg atggggagga ggagggcgag agcgatggca
     1201 cccacccggt gggaaccccc ccagatagcc ccgggaatac actgaacagc tcaccacccc
     1261 tggctcccgg ggagttcgat ccgaacatcg acgtctatgg aattaagtgc catgaaaact
     1321 cgcctaggaa ggaggtgtac ttcatggcaa ttattgacat ccttactcat tatgatgcaa
     1381 aaaagaaagc tgcccatgct gcaaaaactg ttaaacatgg cgctggcgcg gagatctcca
     1441 ccgtgaaccc agaacagtat tcaaagcgct ttttggactt tattggccac atcttgtgac
     1501 agtaaaggtg gatacggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga
     1561 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     1621 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1681 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     1741 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1801 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1861 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1921 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1981 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2041 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2101 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2161 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2221 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     2281 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2341 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2401 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2461 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2521 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     2581 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2641 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2701 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2761 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2821 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2881 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2941 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     3001 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     3061 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3121 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3181 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3241 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3301 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3361 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3421 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3481 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3541 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3601 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3661 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3721 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3781 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3841 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3901 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3961 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     4021 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4081 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     4141 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4201 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4261 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     4321 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     4381 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     4441 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     4501 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     4561 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     4621 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     4681 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     4741 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4801 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4861 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4921 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4981 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     5041 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     5101 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5161 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     5221 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5281 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5341 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5401 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5461 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     5521 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5581 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5641 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5701 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5761 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5821 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5881 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5941 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     6001 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     6061 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     6121 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     6181 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     6241 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6301 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6361 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6421 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg c
//
