LOCUS       sequence-464119-        6125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of DELTA-actitoxin-Afr1a 
            (DELTA-AITX-Afr1a) (Fragaceatoxin C) (fraC) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6125)
  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_4WDC
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..27)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     218..237
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(361..408)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     404..422
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(475..492)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1345..1374)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1397..1419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1434..1458
                     /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(1458..1477)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1459..1477)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1565..1584)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1790..1867
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1868..2950
                     /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(1887..1906)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3759..3950
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3944..3966)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4052..4191
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4107..4126
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4206..4223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4377..4965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4457..4476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5076..5095)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5087..5902
                     /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             5087..5902
                     /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(5160..5179)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5994..6125,1..324))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
       61 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      121 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      181 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      241 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      301 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
      361 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
      421 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg
      481 gtggtggtgg tgctcgagtc attacgaacc cgctttggtg acgtggattt ccagaatagc
      541 atgaccggag ctgttcatga agccacggct cttcagacca taacccaagc cacgggaatg
      601 ccagccatta tcgccgcgaa atgggcttct gtggtagtac agctcttcgt acatacgctg
      661 atccgcgcgc ttttgacctt tataaacacg aacgttccac caattgctgt accaattata
      721 gtcatacgga acgctaaaca gcaccgccag cgtgttaccg tcgctcatcg agtaggcaat
      781 aacacccacc acaccggtgg ccaccgggcc acggttcttc tggccgttat acagcagtgc
      841 cttgccgtgc gcgaccttgt gcggtaacac gatgtcggaa gttccagagc ggaagtaggt
      901 attcattgcg gtccacgttt taccactctc gttgtcaata cctaccgcga ttttacgttt
      961 cacgttgccc agagcctcca aaacggtctt caaaacgtcc ggacctagcc ccgcaccatc
     1021 tataactgct ccagctactc cagagccgcc aatctgttca cggtgggctt caatgatgtc
     1081 gttgtcttcc atgtccaggt cttccggcgc ttgatctgcc tggatgcgaa taccgtcata
     1141 caggaaacgc aggctatcca tttccttgcc ctgacgtttg gcaaacgctt ccatcaggcg
     1201 acgcagcggc gtggtctttt tgatcttaaa gaaaatttca gagctgccgt cactaacttt
     1261 caggttaata tgggtttccg gtttcacttc cggcttgact tccggctttg cttcttggtt
     1321 gacttccgaa tcttgcaggc taccgtggtg atggtgatgg tgatgatggt gatgaatctt
     1381 cgccatcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     1441 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatcgatc
     1501 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     1561 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1621 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1681 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     1741 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     1801 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1861 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1921 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1981 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     2041 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     2101 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     2161 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     2221 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     2281 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     2341 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     2401 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     2461 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     2521 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     2581 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     2641 cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg ggatacgacg ataccgaaga
     2701 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     2761 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     2821 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2881 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2941 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     3001 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     3061 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     3121 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     3181 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     3241 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     3301 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     3361 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     3421 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     3481 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     3541 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3601 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3661 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     3721 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     3781 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     3841 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     3901 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3961 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4021 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     4081 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4141 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     4201 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4261 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4321 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4381 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4441 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4501 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4561 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4621 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4681 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4741 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4801 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4861 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4921 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4981 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5041 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     5101 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     5161 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     5221 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     5281 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     5341 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     5401 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     5461 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     5521 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     5581 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5641 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5701 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5761 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5821 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5881 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     5941 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     6001 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6061 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6121 gagtg
//
