LOCUS       sequence-464139-        6176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of envoplakin in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6176)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6176)
  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_4QMD
FEATURES             Location/Qualifiers
     source          1..6176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(52..99)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     95..113
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(166..183)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1087..1116)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1139..1161
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1176..1200
                     /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(1200..1219)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1201..1219)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1307..1326)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1532..1609
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1610..2692
                     /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(1629..1648)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3501..3692
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3686..3708)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3794..3933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3849..3868
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3948..3965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4119..4707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4199..4218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4818..4837)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4829..5644
                     /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             4829..5644
                     /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(4902..4921)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5736..6176,1..15))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5873..5894)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6085..6104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc
       61 cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc
      121 agccaactca gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg
      181 gtgctcgagt cattacgaac ccgctgccgg aagaagcaac agaccgctca acgggtcttt
      241 acgacaacga cccatagcct ctttgtacga caaacgctct ttgctgatcg ggtccgtcag
      301 gtccttttca tagctagact catcctgcag cagttgcgcc agttcttcgc tgatcatgcc
      361 ggacaacagc gcctgctgga tcggaatacg gccggtgcgt ttcggatcaa tcaggccacc
      421 cgtcaggtgc tgtacttgca gatgtggcag gacggactca cgcggcatcc aacccttttg
      481 aaccgcttca cccacggaga ggcgcttttt ggtaaccgga tcctcgatgc cggtgaacgc
      541 cttttgtgca ttcaacaatc tctgggtgct ggtgttctcg atcagaccac gttccatggc
      601 tttgtgcacg ctgtagcgct cacgagagag cagatcaaca atgccgccgg tagccgcttg
      661 agcttctaac agcttctgac cggtgatagg atccagcatg tttttggcca ctgcggtctt
      721 aatgctgcac ttgttgtccg tagtggtgtc ataaattcca gctatgggaa atgaatcgtc
      781 tccagagccg ccaatctgtt cacggtgggc ttcaatgatg tcgttgtctt ccatgtccag
      841 gtcttccggc gcttgatctg cctggatgcg aataccgtca tacaggaaac gcaggctatc
      901 catttccttg ccctgacgtt tggcaaacgc ttccatcagg cgacgcagcg gcgtggtctt
      961 tttgatctta aagaaaattt cagagctgcc gtcactaact ttcaggttaa tatgggtttc
     1021 cggtttcact tccggcttga cttccggctt tgcttcttgg ttgacttccg aatcttgcag
     1081 gctaccgtgg tgatggtgat ggtgatgatg gtgatgaatc ttcgccatca tatgtatatc
     1141 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     1201 cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct ctacgccgga
     1261 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1321 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     1381 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     1441 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     1501 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1561 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1621 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1681 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1741 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     1801 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     1861 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     1921 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     1981 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     2041 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     2101 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     2161 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     2221 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2281 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2341 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2401 tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     2461 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2521 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2581 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2641 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     2701 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     2761 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2821 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     2881 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     2941 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     3001 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     3061 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     3121 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     3181 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3241 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3301 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3361 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3421 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     3481 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3541 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3601 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3661 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3721 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3781 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     3841 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     3901 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3961 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4021 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc
     4801 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
     4861 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     4921 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     4981 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     5041 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     5101 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     5161 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     5221 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     5281 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     5341 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     5401 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     5461 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     5521 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     5581 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     5641 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5701 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     5761 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5821 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     5881 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5941 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6001 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6061 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6121 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgc
//
