LOCUS       sequence-133184-        6554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic expression of APETx2 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6554)
  AUTHORS   Anangi R, Rash LD, Mobli M, King GF
  TITLE     Functional expression in Escherichia coli of the disulfide-rich sea 
            anemone peptide APETx2, a potent blocker of acid-sensing ion channel
            3.
  JOURNAL   Mar Drugs. 2012 Jul;10(7):1605-18. doi: 10.3390/md10071605. Epub 
            2012 Jul 23.
  PUBMED    22851929
REFERENCE   2  (bases 1 to 6554)
  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"; doi: 
            "10.3390/md10071605"; journalName: "Mar Drugs"; date: "2012-07"; 
            volume: "10"; issue: "7"; pages: "1605-18"
COMMENT     Sequence Label: pLicC-MBP-APETx2
FEATURES             Location/Qualifiers
     source          1..6554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        285..388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(389..457,458..1249)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(607..626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1420..2008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1909..1928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2162..2179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2194..2336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2262..2281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2422..2444
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2438..2629)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3438..4520)
                     /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     4482..4501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4521..4598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4804..4823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4911..4930
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4911..4929
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4930..4954
                     /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             4969..4991
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5083..5100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      6049..6058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6166..6189
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6214..6234
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6398..6415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6468..6486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6482..6529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      361 tgcttcaata atttgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      901 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1081 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2221 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2281 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2341 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2401 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2461 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2521 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2581 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2641 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2701 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2761 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2821 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2881 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2941 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3001 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     3061 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     3121 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     3181 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     3241 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     3301 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     3361 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     3421 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3481 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     3541 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     3601 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     3661 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     3721 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     3781 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     3841 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     3901 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     3961 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4021 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4081 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     4141 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     4201 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     4261 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4321 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4381 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     4441 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     4501 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     4561 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     4621 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     4681 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     4741 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     4801 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     4861 cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact
     4921 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     4981 taagaaggag atatacatat gaaaataaaa acaggtgcac gcatcctcgc attatccgca
     5041 ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa tccatcacca tcaccatcac
     5101 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     5161 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     5221 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     5281 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     5341 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     5401 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     5461 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     5521 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     5581 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     5641 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     5701 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     5761 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     5821 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     5881 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     5941 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     6001 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc caccatggaa
     6061 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     6121 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     6181 gacgcgcaga ctgattacga tatcccaggt accgaaaatc tgtattttca gggcaccgca
     6241 tgtagctgtg gtaatagcaa aggcatttat tggttttatc gtccgagctg tccgaccgat
     6301 cgtggttata ccggtagctg tcgttatttt ctgggcacct gttgtacacc ggcagattaa
     6361 taagagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     6421 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     6481 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     6541 aactatatcc ggat
//
