LOCUS       sequence-464176-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of flagellar motor switch protein 
            FliM in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5969)
  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_1U8T
FEATURES             Location/Qualifiers
     source          1..5969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..105)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     296..315
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(439..486)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     482..500
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(553..570)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1267..1296)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1319..1341
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1356..1380
                     /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(1380..1399)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1381..1399)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1487..1506)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1712..1789
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1790..2872
                     /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(1809..1828)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3681..3872
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3866..3888)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3974..4113
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4029..4048
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4128..4145)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4299..4887)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4379..4398)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4998..5017)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5009..5824
                     /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             5009..5824
                     /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(5082..5101)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5916..5969,1..402))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
       61 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      121 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      181 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      241 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      301 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      361 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga
      421 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
      481 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
      541 agccggatct cagtggtggt ggtggtggtg ctcgagtcat tacgaacctt caaagatctt
      601 gttcagtttt tcttccagcg tagccgcagt aaacggtttt acaacccaac cgctcgcacc
      661 cgcctgcgcc gcggcgataa tgttctcctt tttagcttct gcggtgacca tcagaaccgg
      721 taatgcgctc atggcaccat ctgcgcgaat ggtcttcaac aactccaggc cgtccatatt
      781 cggcatattc caatcagaaa tcacgaagcc gtaaccgccc gcttgcagct tgttcaaagc
      841 atcaacgccg tcctccgcct cttccacgtt gttgaaaccg agctccttca gcagattacg
      901 cacgatacga cgcatggtgg agaatttgtc tacaactaaa aactttagtt ctttatcagc
      961 tccagagccg ccaatctgtt cacggtgggc ttcaatgatg tcgttgtctt ccatgtccag
     1021 gtcttccggc gcttgatctg cctggatgcg aataccgtca tacaggaaac gcaggctatc
     1081 catttccttg ccctgacgtt tggcaaacgc ttccatcagg cgacgcagcg gcgtggtctt
     1141 tttgatctta aagaaaattt cagagctgcc gtcactaact ttcaggttaa tatgggtttc
     1201 cggtttcact tccggcttga cttccggctt tgcttcttgg ttgacttccg aatcttgcag
     1261 gctaccgtgg tgatggtgat ggtgatgatg gtgatgaatc ttcgccatca tatgtatatc
     1321 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     1381 cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct ctacgccgga
     1441 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1501 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     1561 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     1621 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     1681 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1741 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1801 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1861 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1921 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     1981 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     2041 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     2101 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     2161 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     2221 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     2281 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     2341 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     2401 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2461 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2521 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2581 tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     2641 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2701 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2761 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2821 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     2881 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     2941 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     3001 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     3061 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     3121 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     3181 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     3241 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     3301 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     3361 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3421 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3481 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3541 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3601 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     3661 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3721 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3781 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3841 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3901 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3961 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     4021 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     4081 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     4141 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4201 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4261 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4321 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4381 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4441 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4501 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4561 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4621 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4681 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4741 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4801 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4861 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4921 gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc
     4981 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
     5041 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     5101 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     5161 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     5221 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     5281 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     5341 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     5401 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     5461 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     5521 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     5581 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     5641 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     5701 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     5761 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     5821 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5881 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     5941 aaaattcgcg ttaaattttt gttaaatca
//
