LOCUS       sequence-464137-        6026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of ASCH domain-containing 
            ribonuclease in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6026)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6026)
  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_5GUQ
FEATURES             Location/Qualifiers
     source          1..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(127..174)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     170..188
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(241..258)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1012..1041)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1064..1086
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1101..1125
                     /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(1125..1144)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1126..1144)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1232..1251)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1457..1534
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1535..2617
                     /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(1554..1573)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3426..3617
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3611..3633)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3719..3858
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3774..3793
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3873..3890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4044..4632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4124..4143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4743..4762)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4754..5569
                     /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             4754..5569
                     /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(4827..4846)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5661..6026,1..90))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5798..5819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(6010..6026,1..3)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
       61 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata tagttcctcc
      121 tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg
      181 ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag ccggatctca
      241 gtggtggtgg tggtggtgct cgagtcatta cgaaccctca acctgagagg tcaggctaac
      301 cagtttctgc gtttcctctt tgcgcagcca ggttaaggac tgtggcggtt gaaagtttgc
      361 tctgatctcc ttaagttgat ccaggttgat agggcggatc ggctggtgat cacggagcaa
      421 gaaagcggtc gccttttcag taccttcgaa gtagtcacga tattgctgtt ccgacaagaa
      481 ggcttcacgg ccgtagcggc tccacagggt attcacgtca ccttggacaa tcgcctccag
      541 gtaggcaaag ccaatcacgc tttttaccgg acgcgtagca taaatccaga tacgcgcgct
      601 cagtgccggc agcggaatac gacggcggaa ctcaacggtc tttttaccgg acacgatcgc
      661 cttcgcaaac tccggccata gtgatattac agcttctttc ctatctccag agccgccaat
      721 ctgttcacgg tgggcttcaa tgatgtcgtt gtcttccatg tccaggtctt ccggcgcttg
      781 atctgcctgg atgcgaatac cgtcatacag gaaacgcagg ctatccattt ccttgccctg
      841 acgtttggca aacgcttcca tcaggcgacg cagcggcgtg gtctttttga tcttaaagaa
      901 aatttcagag ctgccgtcac taactttcag gttaatatgg gtttccggtt tcacttccgg
      961 cttgacttcc ggctttgctt cttggttgac ttccgaatct tgcaggctac cgtggtgatg
     1021 gtgatggtga tgatggtgat gaatcttcgc catcatatgt atatctcctt cttaaagtta
     1081 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     1141 attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc
     1201 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     1261 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     1321 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     1381 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     1441 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     1501 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     1561 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     1621 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     1681 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     1741 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     1801 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     1861 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     1921 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     1981 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     2041 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     2101 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     2161 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     2221 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     2281 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc
     2341 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     2401 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     2461 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     2521 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     2581 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     2641 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     2701 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     2761 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     2821 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     2881 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     2941 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     3001 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     3061 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     3121 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     3181 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     3241 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     3301 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     3361 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     3421 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     3481 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     3541 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     3601 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3661 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     3721 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     3781 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     3841 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3901 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3961 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4021 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4081 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4141 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4201 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4261 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4321 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4381 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4441 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4501 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4561 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4621 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4681 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     4741 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     4801 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     4861 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     4921 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     4981 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5041 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     5101 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     5161 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     5221 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     5281 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     5341 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     5401 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     5461 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     5521 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     5581 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5641 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5701 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5761 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5821 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5881 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5941 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     6001 gagaaaggaa gggaagaaag cgaaag
//
