LOCUS       sequence-464112-        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of internalin B (InlB) in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6470)
  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_1H6T
FEATURES             Location/Qualifiers
     source          1..6470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     638..657
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     722..741
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      852..1440
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1341..1360
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1594..1611
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1626..1765
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1691..1710)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1851..1873
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1867..2058)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2867..3949)
                     /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"
     promoter        complement(3950..4027)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4233..4252
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4340..4359
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4340..4358
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4359..4383
                     /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             4398..4420
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4443..4472
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5670..5687
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5740..5758)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5754..5801
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5838..6293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5925..5944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6135..6156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6385..6470,1..730))
                     /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(join(6385..6470,1..730))
                     /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"
ORIGIN
        1 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
       61 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
      121 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
      181 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
      241 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
      301 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
      361 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
      421 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
      481 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
      541 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
      601 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
      661 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
      721 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
      781 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
      841 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      901 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      961 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1021 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1081 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1141 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1201 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1261 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1321 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1381 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1441 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1501 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1561 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1621 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     1681 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     1741 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     1801 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     1861 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     1921 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     1981 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     2041 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2101 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2161 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     2221 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     2281 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     2341 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     2401 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     2461 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     2521 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     2581 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     2641 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     2701 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     2761 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     2821 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     2881 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2941 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3001 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3061 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3121 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     3181 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3241 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3301 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3361 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3421 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3481 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3541 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     3601 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     3661 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     3721 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3781 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     3841 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     3901 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     3961 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4021 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     4081 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     4141 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4201 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     4261 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     4321 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     4381 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg atggcgaaga
     4441 ttcatcacca tcatcaccat caccatcacc acggtagcct gcaagattcg gaagtcaacc
     4501 aagaagcaaa gccggaagtc aagccggaag tgaaaccgga aacccatatt aacctgaaag
     4561 ttagtgacgg cagctctgaa attttcttta agatcaaaaa gaccacgccg ctgcgtcgcc
     4621 tgatggaagc gtttgccaaa cgtcagggca aggaaatgga tagcctgcgt ttcctgtatg
     4681 acggtattcg catccaggca gatcaagcgc cggaagacct ggacatggaa gacaacgaca
     4741 tcattgaagc ccaccgtgaa cagattggcg gctctggagg gcccctagga tcagaaacaa
     4801 taactgtacc gaccccgatc aaacagattt tctccgatga cgcgtttgca gagacaatta
     4861 aagacaacct gaaaaaaaag tcggtgacgg acgcggtcac tcaaaacgag ctgaacagca
     4921 ttgatcagat tatcgccaac aactcggaca tcaagagcgt ccagggtatt caatatctgc
     4981 cgaacgtgac caaattattc ctgaatggta acaagttgac cgacatcaaa ccgctggcta
     5041 atctgaagaa tctcggctgg ctgtttttag atgaaaacaa ggtgaaggat ctgtcaagcc
     5101 tgaaggactt gaagaagttg aagtctttgt ccctggagca caacggcatc tctgacatca
     5161 acggtctggt tcatctgcct cagctggaga gcctctacct gggcaataac aaaattaccg
     5221 atatcacggt gttgtctcgc ctgaccaaac tggatactct gagcctggaa gataatcaga
     5281 tcagcgacat cgtgccatta gcgggcttga ccaaacttca gaatttatac ctttccaaaa
     5341 accacatctc cgacctgcgt gcactggctg gtctgaagaa cctggatgtt ctggaactct
     5401 tctctcaaga gtgcctgaat aagccgatta accaccaaag caatctggta gttccgaaca
     5461 ccgttaagaa caccgacggc agcttggtga cgccggagat cattagcgac gatggtgatt
     5521 acgaaaaacc gaatgtgaag tggcatctgc cggagttcac caatgaagtt agctttatct
     5581 tctatcagcc ggttaccatt ggtaaagcga aagcgcgttt tcacggtcgt gttacccaac
     5641 cgctgaaaga aggttcgtaa tgactcgagc accaccacca ccaccactga gatccggctg
     5701 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     5761 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     5821 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5881 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5941 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6001 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6061 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6121 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6181 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6241 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     6301 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6361 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     6421 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
//
