LOCUS       sequence-464185-        5921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of chagasin in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5921)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5921)
  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_2H7W
FEATURES             Location/Qualifiers
     source          1..5921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(108..129)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     320..339
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(463..510)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     506..524
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(577..594)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1243..1272)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1295..1317
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1332..1356
                     /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(1356..1375)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1357..1375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1463..1482)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1688..1765
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1766..2848
                     /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(1785..1804)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3657..3848
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3842..3864)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3950..4089
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4005..4024
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4104..4121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4275..4863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4355..4374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4974..4993)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4985..5800
                     /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             4985..5800
                     /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(5058..5077)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5892..5921,1..426))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
       61 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
      121 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
      181 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
      241 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
      301 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
      361 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
      421 gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac
      481 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc
      541 agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag
      601 tcattacgaa cccgccttca ggtacacgat aaagcgctca gaatcgtggg acggaccggt
      661 ccacggacgc atataggtca ggttcaccgc atgcgtacct gccgctttaa cggtaacatg
      721 aaagtgctca gtgccgcctg cacccagcaa tttgctgtct ggcgggaagt acttgttctc
      781 gacggtaaac atgctttcat tcggcgattc tttcgtaccg ccctcgaaat accaggcgaa
      841 gccggtggtc gggttgctcg ggagctgaat ttcaaccaat tcaccaaccg ccaccgtcag
      901 ggtagctcca ttgtgagcct ttgttacttt atgtgatcca gagccgccaa tctgttcacg
      961 gtgggcttca atgatgtcgt tgtcttccat gtccaggtct tccggcgctt gatctgcctg
     1021 gatgcgaata ccgtcataca ggaaacgcag gctatccatt tccttgccct gacgtttggc
     1081 aaacgcttcc atcaggcgac gcagcggcgt ggtctttttg atcttaaaga aaatttcaga
     1141 gctgccgtca ctaactttca ggttaatatg ggtttccggt ttcacttccg gcttgacttc
     1201 cggctttgct tcttggttga cttccgaatc ttgcaggcta ccgtggtgat ggtgatggtg
     1261 atgatggtga tgaatcttcg ccatcatatg tatatctcct tcttaaagtt aaacaaaatt
     1321 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1381 gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1441 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1501 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1561 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1621 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1681 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1741 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1801 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1861 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1921 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1981 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     2041 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     2101 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     2161 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2221 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2281 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2341 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2401 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2461 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2521 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct cggtagtggg
     2581 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2641 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2701 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2761 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2821 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     2881 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2941 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     3001 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     3061 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     3121 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3181 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3241 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3301 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3361 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3421 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3481 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3541 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3601 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3661 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     3721 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3781 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3841 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3901 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3961 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     4021 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4081 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4681 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4921 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     4981 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     5041 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     5101 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     5161 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     5221 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     5281 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     5341 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     5401 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     5461 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     5521 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     5581 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     5641 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     5701 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     5761 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat
     5821 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5881 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa t
//
