LOCUS       sequence-464175-        5909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of PI3K-C2-gamma in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5909)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5909)
  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: pAIP4_2WWE
FEATURES             Location/Qualifiers
     source          1..5909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..135)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     326..345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(469..516)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     512..530
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(583..600)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1237..1266)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1289..1311
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1326..1350
                     /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)."
     primer_bind     complement(1350..1369)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1351..1369)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1457..1476)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1682..1759
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1760..2842
                     /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     complement(1779..1798)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3651..3842
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3836..3858)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3944..4083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3999..4018
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4098..4115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4269..4857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4349..4368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4968..4987)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4979..5794
                     /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             4979..5794
                     /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     complement(5052..5071)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5886..5909,1..432))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
       61 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
      121 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
      181 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
      241 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
      301 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
      361 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
      421 tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca aaaaacccct
      481 caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg tggcagcagc
      541 caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt ggtggtggtg
      601 ctcgagtcat tacgaaccct cgctcaggaa gaaggacaaa acgcagtcgg aattggtcac
      661 ctcgtggctc acgttcagga tttgttccat ataatgattt aaatcacgaa aacggcggtg
      721 gtcgctgttg gtaaacggca gatgccacca atgcggaaat tccggcaggg tcaagctcgc
      781 aaattgtttc tgcagctgag agtgcagctt gctgaattgc tcgaacgatt tttccgtcaa
      841 tgaagtctcg ttgttggagt gggtaacctg aatcaggtag aggttgctgc tcttcttaga
      901 aaatcctagt attgtagccc tttcaattga tccagagccg ccaatctgtt cacggtgggc
      961 ttcaatgatg tcgttgtctt ccatgtccag gtcttccggc gcttgatctg cctggatgcg
     1021 aataccgtca tacaggaaac gcaggctatc catttccttg ccctgacgtt tggcaaacgc
     1081 ttccatcagg cgacgcagcg gcgtggtctt tttgatctta aagaaaattt cagagctgcc
     1141 gtcactaact ttcaggttaa tatgggtttc cggtttcact tccggcttga cttccggctt
     1201 tgcttcttgg ttgacttccg aatcttgcag gctaccgtgg tgatggtgat ggtgatgatg
     1261 gtgatgaatc ttcgccatca tatgtatatc tccttcttaa agttaaacaa aattatttct
     1321 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     1381 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1441 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1501 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     1561 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     1621 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     1681 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     1741 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1801 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1861 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1921 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1981 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     2041 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     2101 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     2161 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     2221 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     2281 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     2341 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     2401 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     2461 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     2521 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     2581 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     2641 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     2701 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     2761 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2821 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2881 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2941 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     3001 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     3061 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     3121 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     3181 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3241 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3301 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3361 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3421 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3481 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3541 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     3601 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     3661 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     3721 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3781 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3841 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3901 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3961 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     4021 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4081 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4681 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4921 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4981 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     5041 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     5101 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     5161 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     5221 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     5281 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     5341 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     5401 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5461 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5521 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5581 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5641 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5701 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5761 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5821 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5881 cacctaaatt gtaagcgtta atattttgt
//
