LOCUS       sequence-464162-        5978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of aspartate kinase in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5978)
  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_1ZHV
FEATURES             Location/Qualifiers
     source          1..5978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(28..45)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(751..780)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             803..825
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    840..864
                     /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(864..883)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(865..883)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(971..990)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1196..1273
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1274..2356
                     /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(1293..1312)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3165..3356
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3350..3372)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3458..3597
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3513..3532
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3612..3629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3783..4371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3863..3882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4482..4501)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4493..5308
                     /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             4493..5308
                     /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(4566..4585)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5400..5855)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5537..5558)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5749..5768
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5892..5939)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tcgggctttg ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gtcattacga
       61 acccaacaac aggctatgac ccgcattagc caacaggtct gcggtctttt ccagatcgtt
      121 gctacgaacc agtaggtggt caccgtcgaa ggtgctaaca acgaagatgc cgataccgtt
      181 cgtgctcagc gggctgatca cggacagcac gataccagtt tcgtcaaacg caaacggacc
      241 ttggaattta aaacaagacc aacccgggtc cacgcgaaca tcctgtggaa tacgatcaat
      301 caggcagaca atggagagct catcatcggt acgggtgatc gacacgaagc caccgccgtc
      361 agcccatgcc ggaatcgcct cggacgcaga caggcgcgcg ataccgtagc tgccatttaa
      421 tatctttagt ttaatcctgg gtcctccaga gccgccaatc tgttcacggt gggcttcaat
      481 gatgtcgttg tcttccatgt ccaggtcttc cggcgcttga tctgcctgga tgcgaatacc
      541 gtcatacagg aaacgcaggc tatccatttc cttgccctga cgtttggcaa acgcttccat
      601 caggcgacgc agcggcgtgg tctttttgat cttaaagaaa atttcagagc tgccgtcact
      661 aactttcagg ttaatatggg tttccggttt cacttccggc ttgacttccg gctttgcttc
      721 ttggttgact tccgaatctt gcaggctacc gtggtgatgg tgatggtgat gatggtgatg
      781 aatcttcgcc atcatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg
      841 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
      901 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
      961 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1021 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1081 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1141 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     1201 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     1261 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     1321 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     1381 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     1441 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     1501 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     1561 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     1621 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     1681 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     1741 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     1801 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     1861 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     1921 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     1981 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     2041 cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac
     2101 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     2161 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     2221 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     2281 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     2341 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     2401 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     2461 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     2521 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     2581 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     2641 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     2701 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     2761 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     2821 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     2881 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     2941 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     3001 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     3061 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     3121 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     3181 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     3241 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     3301 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     3361 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     3421 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     3481 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     3541 gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3601 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4201 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4381 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4441 gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     4501 tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt
     4561 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     4621 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     4681 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     4741 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     4801 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     4861 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     4921 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     4981 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     5041 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     5101 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     5161 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     5221 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     5281 tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat
     5341 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     5401 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5461 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5521 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5581 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5641 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5701 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5761 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5821 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt
     5881 cctcctttca gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt
     5941 tattgctcag cggtggcagc agccaactca gcttcctt
//
