LOCUS       sequence-464114-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of serine-aspartate 
            repeat-containing protein E in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6539)
  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_5IHW
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    118..257
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(183..202)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     343..365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(359..550)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1359..2441)
                     /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     2403..2422
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2442..2519)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2725..2744
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2832..2851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2832..2850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2851..2875
                     /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             2890..2912
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2935..2964
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4231..4248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4301..4319)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4315..4362
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4399..4854
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4486..4505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4696..4717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4946..5761)
                     /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(4946..5761)
                     /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     5669..5688
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5753..5772
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5883..6471
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6372..6391
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
       61 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
      121 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
      181 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
      241 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      301 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      361 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
      421 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
      481 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
      541 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
      601 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
      661 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
      721 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
      781 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
      841 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
      901 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
      961 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     1021 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     1081 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     1141 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     1201 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     1261 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     1321 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1381 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1441 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     1501 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     1561 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     1621 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     1681 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     1741 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     1801 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     1861 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     1921 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     1981 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     2041 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     2101 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     2161 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     2221 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     2281 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     2341 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     2401 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     2461 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     2521 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     2581 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     2641 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     2701 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     2761 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     2821 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     2881 agaaataatt ttgtttaact ttaagaagga gatatacata tgatggcgaa gattcatcac
     2941 catcatcacc atcaccatca ccacggtagc ctgcaagatt cggaagtcaa ccaagaagca
     3001 aagccggaag tcaagccgga agtgaaaccg gaaacccata ttaacctgaa agttagtgac
     3061 ggcagctctg aaattttctt taagatcaaa aagaccacgc cgctgcgtcg cctgatggaa
     3121 gcgtttgcca aacgtcaggg caaggaaatg gatagcctgc gtttcctgta tgacggtatt
     3181 cgcatccagg cagatcaagc gccggaagac ctggacatgg aagacaacga catcattgaa
     3241 gcccaccgtg aacagattgg cggctctgga aacaatgtta atgatctaat aaaagtaaca
     3301 aagcaaacca tcaaggtggg tgatggcaaa gataatgtag ccgcagctca cgatggtaag
     3361 gacatcgagt atgacacgga atttaccatc gacaacaaag tgaaaaaggg tgataccatg
     3421 accatcaatt acgacaagaa cgtcatcccg agcgatctca cggataaaaa cgacccgatt
     3481 gacatcactg acccaagcgg tgaagttatc gcgaaaggca cgttcgataa ggcgaccaag
     3541 cagattacct ataccttcac cgattacgtt gacaagtacg aggacatcaa atctcgtctg
     3601 accctgtata gctatattga caagaagact gtcccgaatg agacttcatt gaatttgacc
     3661 ttcgccaccg caggtaaaga aacgtctcaa aacgtgacgg tggactatca ggatccgatg
     3721 gtgcatggtg acagcaacat tcagtccatt tttaccaaac tggatgaaga taagcaaacc
     3781 attgagcagc agatttacgt gaacccgctg aaaaagtctg cgacgaatac caaagtggat
     3841 attgctggca gccaggttga cgactacggc aacatcaagc tgggtaatgg ttcgaccatc
     3901 atcgatcaga acaccgaaat taaagtctat aaagttaatt cggaccaaca actgccgcag
     3961 agcaatcgta tctacgactt ctcccaatat gaggatgtta ccagccaatt tgataacaag
     4021 aagagctttt ctaacaacgt ggcgacgtta gatttcggcg acatcaacag cgcgtatatc
     4081 attaaagttg ttagcaaata caccccgacc tccgatggcg agctggacat tgcgcagggt
     4141 acaagcatgc gcaccaccga caagtacggc tactataact acgcaggcta ctccaacttt
     4201 atcgttacca gcggttcgta atgactcgag caccaccacc accaccactg agatccggct
     4261 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     4321 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     4381 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4441 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4501 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4561 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4621 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4681 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4741 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4801 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4861 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4921 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     4981 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     5041 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     5101 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     5161 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     5221 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     5281 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     5341 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     5401 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     5461 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     5521 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     5581 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     5641 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     5701 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     5761 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     5821 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5881 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5941 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6001 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     6061 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6121 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6181 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6241 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6301 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6361 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6421 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6481 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcc
//
