LOCUS       sequence-464129-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of flagellin in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6371)
  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_4CFI
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(116..1198)
                     /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     1160..1179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1199..1276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1482..1501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1589..1608
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1589..1607
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1608..1632
                     /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             1647..1669
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1692..1721
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2820..2837
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2890..2908)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2904..2951
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2988..3443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3075..3094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3285..3306
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3535..4350)
                     /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(3535..4350)
                     /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     4258..4277
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4342..4361
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4472..5060
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4961..4980
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5214..5231
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5246..5385
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5311..5330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5471..5493
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5487..5678)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
       61 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
      121 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      181 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
      241 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
      301 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
      361 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
      421 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
      481 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
      541 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
      601 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
      661 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
      721 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
      781 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
      841 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
      901 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
      961 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     1021 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     1081 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     1141 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     1201 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     1261 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     1321 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     1381 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     1441 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     1501 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     1561 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1621 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatga
     1681 tggcgaagat tcatcaccat catcaccatc accatcacca cggtagcctg caagattcgg
     1741 aagtcaacca agaagcaaag ccggaagtca agccggaagt gaaaccggaa acccatatta
     1801 acctgaaagt tagtgacggc agctctgaaa ttttctttaa gatcaaaaag accacgccgc
     1861 tgcgtcgcct gatggaagcg tttgccaaac gtcagggcaa ggaaatggat agcctgcgtt
     1921 tcctgtatga cggtattcgc atccaggcag atcaagcgcc ggaagacctg gacatggaag
     1981 acaacgacat cattgaagcc caccgtgaac agattggcgg ctctggagat ggagtatcaa
     2041 tactacaaac agctagttct ggtctgacct ctctgacgaa cagccttcaa cgtattcgtc
     2101 agctggccgt acaggcatct aatggtccgc tgagcgcgtc cgatgcaagt gcgttgcaac
     2161 aagaggttgc tcagcagatc agcgaagtta accgtatcgc gagccagacg aactataacg
     2221 gcaagaacat cctggatggt tccgctggca ccttgtcatt ccaagttggt gctaacgtgg
     2281 gtcaaaccgt ttcggttgat ctgacccaaa gcatgagcgc tgcgaaaatt ggtggtggca
     2341 tggttcaaac cggccagacc ttgggcacca ttaaggtggc gattgacagc agcggtgccg
     2401 cgtggagcag cggaagcacc ggccaggaga caacccagat caatgtggtg tccgacggca
     2461 aaggtggctt caccttcacc gaccaaaaca accaggcgct gtcgtccacc gcggtcaccg
     2521 ctgtctttgg ctccagcacg gcgggtactg gtactgcggc atctccgagc tttcagacgt
     2581 tagctttgtc caccagcgcg accagcgcgc tctctgcgac cgaccaggcg aatgctactg
     2641 ctatggtggc ccaaatcaac gccgttaata aaccgcagac cgtgagcaat ctggacatca
     2701 gcacgcagac cggcgcgtac caggccatgg tgtccattga taatgctctg gcaaccgtca
     2761 ataacctgca agcgacgctg ggtgcagcgc aaaaccgttt tggttcgtaa tgactcgagc
     2821 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     2881 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2941 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3001 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3061 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3121 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3181 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3241 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3301 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3361 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3421 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3481 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3541 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3601 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3661 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3721 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3781 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3841 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3901 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3961 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4021 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     4081 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4141 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4201 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4261 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4321 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4381 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4441 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4501 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4561 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4621 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4681 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4741 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4801 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4861 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4921 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4981 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5041 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5101 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5161 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5221 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5281 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5341 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5401 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5461 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5521 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5581 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5641 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5701 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5761 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5821 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5881 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5941 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     6001 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     6061 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     6121 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     6181 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     6241 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     6301 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     6361 agtgcggcga c
//
