LOCUS       sequence-464117-        6665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of RalF in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6665)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6665)
  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_1XSZ
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1332..1361)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1384..1406
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1421..1445
                     /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(1445..1464)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1446..1464)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1552..1571)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1777..1854
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1855..2937
                     /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(1874..1893)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3746..3937
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3931..3953)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4039..4178
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4094..4113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4193..4210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4364..4952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4444..4463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5063..5082)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5074..5889
                     /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             5074..5889
                     /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(5147..5166)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5981..6436)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6118..6139)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6330..6349
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6473..6520)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6516..6534
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6587..6604)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 tggttgtcgt acgccttaat caggtcgccc acctcgtagt cataggtagc cttgaagttc
       61 ttaacagggc ttgaaaaatc cgccgccgcc ttagccgcca ggtcgatgct ctctttggtc
      121 tgacccggtt ggataataac acgcggctgt tcaccaaaca gccatttgct aaagatattc
      181 ggagtataaa cctggatggt tgcctgtgcg ctggtcttat tgtcagtcag ggtaattgta
      241 cccttgtaac cggtaaagaa gctcagccaa cttttcggct gatccacggt cgtcttgacg
      301 ttgtcgccaa tgcccggaaa aacggtgttg atgttcacgt ccgtgctgtg taagaaggaa
      361 tccagttttt tgaacgtaga atctttgttg agggtggtcg acgtcagctc ataacccgga
      421 gacgtcttga cgaagttcag ttcaaacggt ttcgccttga tttcgctata cagctcttcc
      481 aaaaacttcg catcaaagtc gccgccgtta ttaccaccgc gcaaattacg tttcaggccg
      541 tctacggtca ttttgttttt ctccggaata gacggattgt gcaagtcggt gttcagcata
      601 atggtctgga acgccagaag gtaggctgca tcagcattgc taacaacatc cgggttttgt
      661 tggaagtatg caccggagaa ggactgaacc agacgatcga ttttttgggc ctcgcctggc
      721 agcttgaaag tcttcaagaa ggtgcgcaga ccttccacga agctctggcc gttgaaattc
      781 atctggctgg tgaaggcctt cagcacttgc tgattttccg cttccggcga gctcaggtag
      841 tcacccacag cctccaaatc aaggttttta cgttgctgat gaaaaaattc agcaatctct
      901 tcatttgggg aaattttata ttgttcgcag atctctttga tcttgttgat accgtttttc
      961 ggctttgcgt taaacgcctc gataatttca cgttgtgctt tctctatttc ggggtgtgaa
     1021 gctcctccag agccgccaat ctgttcacgg tgggcttcaa tgatgtcgtt gtcttccatg
     1081 tccaggtctt ccggcgcttg atctgcctgg atgcgaatac cgtcatacag gaaacgcagg
     1141 ctatccattt ccttgccctg acgtttggca aacgcttcca tcaggcgacg cagcggcgtg
     1201 gtctttttga tcttaaagaa aatttcagag ctgccgtcac taactttcag gttaatatgg
     1261 gtttccggtt tcacttccgg cttgacttcc ggctttgctt cttggttgac ttccgaatct
     1321 tgcaggctac cgtggtgatg gtgatggtga tgatggtgat gaatcttcgc catcatatgt
     1381 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     1441 attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg
     1501 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     1561 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     1621 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     1681 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     1741 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     1801 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     1861 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     1921 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     1981 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     2041 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     2101 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     2161 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     2221 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     2281 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     2341 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     2401 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     2461 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     2521 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     2581 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     2641 cgcgttggtg cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     2701 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     2761 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     2821 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2881 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2941 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     3001 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     3061 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     3121 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     3181 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     3241 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     3301 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     3361 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     3421 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     3481 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     3541 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     3601 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     3661 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     3721 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     3781 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     3841 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     3901 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3961 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4021 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     4081 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     4141 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     4201 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4261 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4321 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4381 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4441 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4501 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4561 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4621 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4681 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4741 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4801 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4861 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4921 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4981 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     5041 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     5101 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     5161 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     5221 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     5281 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     5341 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     5401 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     5461 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     5521 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     5581 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     5641 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     5701 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     5761 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     5821 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     5881 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5941 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     6001 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     6061 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     6121 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     6181 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     6241 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6301 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     6361 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     6421 ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac
     6481 ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag
     6541 cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt
     6601 ggtgctcgag tcattacgaa cccgccttta gcgccaagtt gcgctcgatg gtgatcagct
     6661 ttttc
//
