LOCUS       sequence-464142-        6005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of C20G4.08 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6005)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6005)
  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_4Q2S
FEATURES             Location/Qualifiers
     source          1..6005
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(336..1418)
                     /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     1380..1399
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1419..1496)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1702..1721
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1809..1828
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1809..1827
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1828..1852
                     /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             1867..1889
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1912..1941
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2674..2691
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2744..2762)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2758..2805
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2842..3297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2929..2948)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3139..3160
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3389..4204)
                     /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(3389..4204)
                     /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     4112..4131
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4196..4215
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4326..4914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4815..4834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5068..5085
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5100..5239
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5165..5184)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5325..5347
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5341..5532)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
       61 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
      121 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
      181 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
      241 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
      301 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      361 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      421 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      481 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      541 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      601 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      661 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      721 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      781 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      841 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      901 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      961 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1021 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1081 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1141 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1201 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1261 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1321 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1381 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1441 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1501 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1561 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1621 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1681 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1741 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     1801 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1861 aataattttg tttaacttta agaaggagat atacatatga tggcgaagat tcatcaccat
     1921 catcaccatc accatcacca cggtagcctg caagattcgg aagtcaacca agaagcaaag
     1981 ccggaagtca agccggaagt gaaaccggaa acccatatta acctgaaagt tagtgacggc
     2041 agctctgaaa ttttctttaa gatcaaaaag accacgccgc tgcgtcgcct gatggaagcg
     2101 tttgccaaac gtcagggcaa ggaaatggat agcctgcgtt tcctgtatga cggtattcgc
     2161 atccaggcag atcaagcgcc ggaagacctg gacatggaag acaacgacat cattgaagcc
     2221 caccgtgaac agattggcgg ctctggaata gctgaatcac taagaaggtt aaaagagtac
     2281 gtaaaggcgg gtagcgtcaa ggagtgcgtg gccgagtggt gtaatatgcc aagcgtggct
     2341 ggcttcgacg ttctgagcga gatctcctat gatcgtatgc tggaaaactg cagcaatctg
     2401 ttgttgttaa cgtttatcta tcacatttct ctgctggact ctgtcgatga cgaccgcctg
     2461 tcgaaacgta tggaatacat tagccgtatt tgcctgaaca tcgacgtgaa cgatccgaag
     2521 gtggaaaccg ttgtgcatcc ggttctgacc ctgacccgtg aagcgctcct gcgtcagtcc
     2581 gagttcttca gcccgatttt taaacgccgt ttggttgttc ttttgcgcgc actggatggt
     2641 aaaatctccg agatcggttc gtaatgactc gagcaccacc accaccacca ctgagatccg
     2701 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2761 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2821 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2881 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2941 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3001 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3061 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3121 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3181 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3241 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     3301 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3361 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     3421 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3481 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3541 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3601 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3661 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3721 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3781 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3841 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3901 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3961 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     4021 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4081 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4141 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     4201 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     4261 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4321 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4381 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4441 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4501 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4561 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4621 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4681 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4741 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4801 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4861 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4921 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4981 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5041 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5101 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5161 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5221 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5281 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5341 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5401 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5461 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5521 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5581 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5641 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5701 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5761 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5821 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5881 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5941 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     6001 ggccg
//
