LOCUS       sequence-464159-        6095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of lytic chitin monooxygenase in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6095)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6095)
  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_4A02
FEATURES             Location/Qualifiers
     source          1..6095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             786..977
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(971..993)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1079..1218
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1134..1153
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1233..1250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1404..1992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1484..1503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2103..2122)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2114..2929
                     /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             2114..2929
                     /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(2187..2206)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3021..3476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3158..3179)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3370..3389
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3513..3560)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3556..3574
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3627..3644)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4467..4496)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4519..4541
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4556..4580
                     /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(4580..4599)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4581..4599)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4687..4706)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4912..4989
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4990..6072
                     /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(5009..5028)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
       61 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
      121 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
      181 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
      241 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
      301 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
      361 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
      421 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
      481 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
      541 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
      601 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
      661 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
      721 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
      781 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
      841 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
      901 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
      961 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     1021 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     1081 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     1141 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     1201 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1801 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     1861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2041 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     2101 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     2161 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     2221 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     2281 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2341 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2401 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     2461 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     2521 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     2581 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     2641 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     2701 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     2761 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     2821 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     2881 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     2941 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3001 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3061 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3121 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3181 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     3241 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3301 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     3361 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     3421 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     3481 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     3541 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     3601 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag tcattacgaa
     3661 ccctggatgt tcacgtcaat cgcctggtag aatgcattaa cggtatcacc aataccccaa
     3721 actgcataaa taacatgata acccttgcga tcggtcggga tgttaatggt ctgtttcacc
     3781 actttattcg gaacctcttg cttgccgtcg atagaggcga ttttatcaaa gtttttgatg
     3841 tccaacggct ggttcggatt ccagccgttc ttggtgatgt agtaatccca ggacgcggta
     3901 cggtgttgcg cggtcaggtt ccaagtgatg tccagtgggc cggtcgtaat gttggttttg
     3961 tgccaacggg tcgcagtctg ctcgtccagc ggttcaaaac cgctgacacc cgccgatgcc
     4021 agcttgcccg tgataaacgt gttcttcgga gcttcaatgc tttgcggctc ccattgagca
     4081 cgacccacgt tggtattcaa attaccgccg gcgctgctgc cgaagaacgc ccttgacccg
     4141 ggactagcta catatccgtg tccagagccg ccaatctgtt cacggtgggc ttcaatgatg
     4201 tcgttgtctt ccatgtccag gtcttccggc gcttgatctg cctggatgcg aataccgtca
     4261 tacaggaaac gcaggctatc catttccttg ccctgacgtt tggcaaacgc ttccatcagg
     4321 cgacgcagcg gcgtggtctt tttgatctta aagaaaattt cagagctgcc gtcactaact
     4381 ttcaggttaa tatgggtttc cggtttcact tccggcttga cttccggctt tgcttcttgg
     4441 ttgacttccg aatcttgcag gctaccgtgg tgatggtgat ggtgatgatg gtgatgaatc
     4501 ttcgccatca tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     4561 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
     4621 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     4681 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     4741 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     4801 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     4861 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     4921 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4981 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     5041 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     5101 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     5161 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     5221 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     5281 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     5341 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     5401 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     5461 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     5521 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     5581 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     5641 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     5701 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     5761 accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga cgataccgaa
     5821 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5881 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5941 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     6001 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     6061 agcgggcagt gagcgcaacg caattaatgt aagtt
//
