LOCUS       sequence-464158-        5987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of B7LG64_ECO55 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5987)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5987)
  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_5M2Y
FEATURES             Location/Qualifiers
     source          1..5987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(147..194)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     190..208
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(261..278)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(993..1022)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1045..1067
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1082..1106
                     /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(1106..1125)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1107..1125)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1213..1232)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1438..1515
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1516..2598
                     /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(1535..1554)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3407..3598
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3592..3614)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3700..3839
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3755..3774
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3854..3871)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4025..4613)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4105..4124)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4724..4743)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4735..5550
                     /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             4735..5550
                     /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(4808..4827)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5642..5987,1..110))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5779..5800)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
       61 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
      121 aatccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca
      181 aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct
      241 ttgttagcag ccggatctca gtggtggtgg tggtggtgct cgagtcatta cgaaccagtg
      301 cgcagcacag caaacaactc aagtttcacg tcacccaggc tcgctggggt atagaaggta
      361 caacgacccg cgtccagcat tgcgcgagcc gcatccgtcg acaggtcgag ggcgaagtac
      421 tggttctcca agcgcagcgg gatcgcagcc ggcacgtggg taaccggacg gataataaca
      481 ccggacaacg caacatttac gacttcgctc acgtcgtccg gggagcctgc tttgcacagt
      541 tgcgggaact tggtctgtaa ttcatgaccc ggcatgctgc tacgaacaga cagccaaaaa
      601 tccgcgcctt cacgcagacg ggcatcgtgc agcgcgccct cccacatcac acccttttgt
      661 tttagttcta taaaaactac ccttgatcca gagccgccaa tctgttcacg gtgggcttca
      721 atgatgtcgt tgtcttccat gtccaggtct tccggcgctt gatctgcctg gatgcgaata
      781 ccgtcataca ggaaacgcag gctatccatt tccttgccct gacgtttggc aaacgcttcc
      841 atcaggcgac gcagcggcgt ggtctttttg atcttaaaga aaatttcaga gctgccgtca
      901 ctaactttca ggttaatatg ggtttccggt ttcacttccg gcttgacttc cggctttgct
      961 tcttggttga cttccgaatc ttgcaggcta ccgtggtgat ggtgatggtg atgatggtga
     1021 tgaatcttcg ccatcatatg tatatctcct tcttaaagtt aaacaaaatt atttctagag
     1081 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     1141 gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     1201 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     1261 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     1321 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     1381 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     1441 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     1501 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     1561 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     1621 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     1681 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     1741 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     1801 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     1861 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     1921 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     1981 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     2041 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     2101 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     2161 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     2221 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     2281 gccattaccg agtccgggct gcgcgttggt gcggacatct cggtagtggg atacgacgat
     2341 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     2401 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     2461 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     2521 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     2581 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     2641 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     2701 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     2761 ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat
     2821 gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac
     2881 tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc
     2941 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     3001 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     3061 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     3121 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     3181 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
     3241 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     3301 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     3361 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     3421 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     3481 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     3541 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3601 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     3661 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     3721 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     3781 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     3841 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3901 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3961 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4021 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4081 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4141 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4201 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4261 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4321 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4381 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4441 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4501 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4561 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4621 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4681 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4741 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     4801 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     4861 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     4921 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     4981 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5041 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5101 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5161 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5221 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5281 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     5341 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     5401 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     5461 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     5521 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     5581 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5641 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5701 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5761 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5821 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5881 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5941 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaa
//
