LOCUS       sequence-464173-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of response regulator homolog in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5960)
  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_2NT3
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     672..691
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     756..775
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      886..1474
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1375..1394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1628..1645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1660..1799
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1725..1744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1885..1907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1901..2092)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2901..3983)
                     /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     3945..3964
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3984..4061)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4267..4286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4374..4393
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4374..4392
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4393..4417
                     /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             4432..4454
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4477..4506
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5194..5211
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5264..5282)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5278..5325
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5362..5817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5449..5468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5659..5680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5909..5960,1..764))
                     /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(5909..5960,1..764))
                     /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 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
       61 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
      121 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
      181 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
      241 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
      301 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
      361 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
      421 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
      481 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
      541 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
      601 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
      661 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
      721 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
      781 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
      841 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      901 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      961 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1021 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1081 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1141 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1201 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     1261 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     1321 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     1381 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     1441 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     1501 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     1561 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     1621 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     1681 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     1741 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     1801 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     1861 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1921 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     1981 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     2041 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     2101 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     2161 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     2221 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     2281 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     2341 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     2401 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     2461 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     2521 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     2581 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     2641 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     2701 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     2761 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     2821 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     2881 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2941 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     3001 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     3061 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     3121 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     3181 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     3241 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     3301 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     3361 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     3421 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     3481 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     3541 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     3601 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     3661 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     3721 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     3781 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     3841 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     3901 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     3961 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     4021 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     4081 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     4141 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     4201 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     4261 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     4321 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg
     4381 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     4441 ctttaagaag gagatataca tatgatggcg aagattcatc accatcatca ccatcaccat
     4501 caccacggta gcctgcaaga ttcggaagtc aaccaagaag caaagccgga agtcaagccg
     4561 gaagtgaaac cggaaaccca tattaacctg aaagttagtg acggcagctc tgaaattttc
     4621 tttaagatca aaaagaccac gccgctgcgt cgcctgatgg aagcgtttgc caaacgtcag
     4681 ggcaaggaaa tggatagcct gcgtttcctg tatgacggta ttcgcatcca ggcagatcaa
     4741 gcgccggaag acctggacat ggaagacaac gacatcattg aagcccaccg tgaacagatt
     4801 ggcggctctg gaaagaaaat tctaatagta gaatcagata cagcgttgag cgctaccctg
     4861 cgctccgcgt tagaaggtcg cggtttcacc gtcgacgaaa ccacggatgg taagggctct
     4921 gttgagcaga ttcgtcgtga tcgtccagat ctggtagttc tggctgtgga tctgagcgcg
     4981 ggtcagaacg gttacctgat ctgcggtaaa ctcaagaaag atgacgacct gaagaacgtt
     5041 ccgattgtga tcattggcaa tccggacggc ttcgcccaac atcgtaaatt gaaggcccac
     5101 gcggacgagg cagttgccaa accggtggac gcggatcaat tggtggagag agcgggcgca
     5161 ctgatcggct ttccgggttc gtaatgactc gagcaccacc accaccacca ctgagatccg
     5221 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     5281 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     5341 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5401 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5461 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5521 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5581 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5641 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5701 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5761 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     5821 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5881 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     5941 gcaatttatt catatcagga
//
