LOCUS       sequence-464109-        6728 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Heterologous protein expression of unsaturated glucuronyl hydrolase 
            in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6728)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAIP4_2D5J
FEATURES             Location/Qualifiers
     source          1..6728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1420..1449)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1472..1494
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1509..1533
                     /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(1533..1552)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1534..1552)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1640..1659)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1865..1942
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1943..3025
                     /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(1962..1981)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3834..4025
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4019..4041)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4127..4266
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4182..4201
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4281..4298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4452..5040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4532..4551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5151..5170)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5162..5977
                     /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             5162..5977
                     /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(5235..5254)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6069..6524)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6206..6227)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6418..6437
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6561..6608)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6604..6622
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6675..6692)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 atagcccgtc acaccgcgct ccaggcgcag cagagcttcc aggtagtagt agtcgcccca
       61 aatggtgtag tcatccgggc taatcccgcc tctaacgtgg taggagccac gacggatgaa
      121 accttccgct tcgccgtcgt cgcgctctgc gtagccgtcc ctcaacgcgg tgacggtggt
      181 cttcgccgcg tcgataaaac gttggcgctc tggatcagat tcgtccagtt gcgacgcaat
      241 ttccaacaga ccgcacgcag taatggctga cgcggagcta tcgcggtagc tgctcggttc
      301 ttgcggaact tcaaaatccc aatacaccac gccatcttcc gggacacgcg ccagaaaatg
      361 acgagccata cgtttagctg tctcgagcag atccgcgtta cccagataac ggctgtttaa
      421 ggcgaaaccg tagatacccc aggcctggcc acgcgtccag gtggaaccgt ccgtgttacc
      481 ctggtgggtg ccaccacgga tcgcattacc gttctccgga tcgaaataaa aggtgtggta
      541 gctgctatcg tcaccacgaa ccaagaaacg acgagacttc aatgcatgcg cctcggccac
      601 gcggcggtac tccgggtcac cggtctgctc acctgcccac agtaacagcg gcagattcaa
      661 cagacaatcg atgataatac gaccgccgtt ctcagggtca cctttcggac cccaagcctg
      721 gatgataccg gcgtctgcac gccagcgacg catcagcaca tctgccgcgt ccaaagccag
      781 tttgcgagcg gactcatctt tttccacaat ccactgcgcc ttcgcgctca ggctataaag
      841 gaaaccaata tcgtggtggt ccaggttctc gaaacgatcc agacgctcac gaaaagaggc
      901 aacggtacga actgcgcctt cacgatattg ctcgtcaccg gtgtattcat agcacagcca
      961 gagaatgccg ctccagaaac catcggtcca gtcagtatta tcgttcagaa catacttatt
     1021 agagccgtcg cttacatgcg gaaagcggtc gccgaatttc ttcaagttac gcgcggtgat
     1081 gcctagtgca tctcctatag cttgctgcca cattccagag ccgccaatct gttcacggtg
     1141 ggcttcaatg atgtcgttgt cttccatgtc caggtcttcc ggcgcttgat ctgcctggat
     1201 gcgaataccg tcatacagga aacgcaggct atccatttcc ttgccctgac gtttggcaaa
     1261 cgcttccatc aggcgacgca gcggcgtggt ctttttgatc ttaaagaaaa tttcagagct
     1321 gccgtcacta actttcaggt taatatgggt ttccggtttc acttccggct tgacttccgg
     1381 ctttgcttct tggttgactt ccgaatcttg caggctaccg tggtgatggt gatggtgatg
     1441 atggtgatga atcttcgcca tcatatgtat atctccttct taaagttaaa caaaattatt
     1501 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     1561 ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     1621 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     1681 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     1741 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     1801 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
     1861 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     1921 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     1981 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     2041 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     2101 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     2161 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     2221 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     2281 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     2341 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     2401 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     2461 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     2521 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     2581 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     2641 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     2701 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata
     2761 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     2821 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     2881 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     2941 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     3001 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     3061 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     3121 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     3181 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     3241 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     3301 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     3361 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     3421 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     3481 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     3541 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     3601 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     3661 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     3721 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     3781 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     3841 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     3901 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     3961 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     4021 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     4081 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     4141 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     4201 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4261 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4321 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4381 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4441 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4501 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4561 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4621 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4681 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4741 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4801 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4861 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     4921 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4981 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5041 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5101 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     5161 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     5221 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     5281 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     5341 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     5401 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     5461 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     5521 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     5581 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     5641 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     5701 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     5761 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     5821 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     5881 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     5941 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     6001 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6061 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     6121 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     6181 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     6241 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     6301 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     6361 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     6421 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     6481 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg
     6541 gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt
     6601 tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta
     6661 gcagccggat ctcagtggtg gtggtggtgg tgctcgagtc attacgaacc acggcctctt
     6721 tcgtacca
//
