LOCUS       sequence-394481-        8003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and characterization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8003)
  AUTHORS   Chen Z, Zakrzewska S, Hajare HS, Alvarez-Buylla A, Abderemane-Ali F,
            Bogan M, Ramirez D, O'Connell LA, Du Bois J, Minor DL Jr
  TITLE     Definition of a saxitoxin (STX) binding code enables discovery and 
            characterization of the anuran saxiphilin family.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Nov;119(44):e2210114119. doi: 
            10.1073/pnas.2210114119. Epub 2022 Oct 24.
  PUBMED    36279441
REFERENCE   2  (bases 1 to 8003)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-11"; volume: "119(44):e2210114119. 
            doi"; pages: " 10.1073/pnas.2210114119. Epub 2022 Oct 24"
COMMENT     Sequence Label: RcSxph Y558A/I782A
FEATURES             Location/Qualifiers
     source          1..8003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        156..247
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     172..189
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2834..2857
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2873..2875,2876..3586)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2915..2936)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3176..3195)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3523..3544
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3599..3628
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    3764..3898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3801..3820)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3855..3874
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3927..4092
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4276..4730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4363..4382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4572..4593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4756..4860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4861..4929,4930..5721)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5079..5098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5892..6480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6381..6400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6634..6651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6785..7009
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(7076..7609)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7779..7784,7785..7801,7802..7807))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc
       61 atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc aggactctag ctatagttct
      121 agtggttggc tacgtatact ccggaatatt aatagatcat ggagataatt aaaatgataa
      181 ccatctcgca aataaataag tattttactg ttttcgtaac agttttgtaa taaaaaaacc
      241 tataaatatt ccggattatt cataccgtcc caccatcggg cgcggatcca tggctcctac
      301 cttccagacc gctctgttct tcaccatcat ctccctgtcc ttcgctatgg ccccgaacgc
      361 aaagcaagtc cgctggtgtg ctatcagcga cctggagcag aagaaatgca acgatctggt
      421 tggtagctgc aacgtgccgg acatcaccct ggtgtgcgtt ctgcgtagca gcaccgaaga
      481 ttgcatgacc gcgatcaagg acggtcaggc ggatgctatg ttcctggaca gcggcgaggt
      541 gtacgaagcg agcaaagatc cgtacaacct gaagccgatc atcgctgaac cgtacagcag
      601 caaccgtgac ctgcagaagt gcctgaaaga gcgtcagcag gcgctggcta agaaaatgat
      661 cggtcactac atcccgcagt gcgatgagaa aggcaactac caaccgcagc agtgccacgg
      721 tagcaccggt cactgctggt gcgttaacgc gatgggtgaa aagatcagcg gtaccaacac
      781 cccgccgggt cagacccgtg ctacctgcga gcgtcacgaa ctgccgaagt gcctgaaaga
      841 acgtcaggtg gcgctgggtg gcgacgagaa agttctgggt cgtttcgtgc cgcagtgcga
      901 tgaaaagggc aactacgagc cgcagcagtt ccacggtagc accggctact cctggtgtgt
      961 taacgcgatc ggcgaggaaa tcgctggcac caaaaccccg ccgggcaaga tcccggctac
     1021 ctgccagaag cacgacctgg tgaccacctg ccactacgcg gttgctatgg tgaagaaaag
     1081 cagcgcgttc cagttcaacc agctgaaggg caaacgtagc tgccacagcg gtgttagcaa
     1141 aaccgacggc tggaaggcgc tggtgaccgt tctggtggaa aagaaactgc tgagctggga
     1201 tggtccggcg aaagagagca tccagcgtgc tatgagcaag ttcttcagcg tgagctgcat
     1261 tccgggtgct acccagacca acctgtgcaa gcagtgcaaa ggcgaggaag gcaagaactg
     1321 caaaaacagc cacgacgaac cgtactacgg taactacggc gcgttccgtt gcctgaagga
     1381 agacatgggt gacgttgcgt tcctgcgtag caccgctctg agcgacgagc acagcgaagt
     1441 gtacgagctg ctgtgcccgg ataacacccg taagccgctg aacaagtaca aagagtgcaa
     1501 cctgggtacc gttccggctg gcaccgtggt tacccgtaag atcagcgaca aaaccgaaga
     1561 catcaacaac ttcctgatgg aggcgcagaa gcgtcagtgc aaactgttca gcagcgctca
     1621 cggcaaggac ctgatgttcg acgatagcac cctgcagctg gctctgctga gcagcgaggt
     1681 tgatgctttc ctgtacctgg gtgtgaaact gttccacgcg atgaaggctc tgaccggcga
     1741 cgctcacctg ccgagcaaga acaaagtgcg ttggtgcacc atcaacaagc tggaaaagat
     1801 gaaatgcgac gattggagcg cggttagcgg tggcgcgatc gcttgcaccg aggctagctg
     1861 cccgaagggc tgcgtgaaac agatcctgaa gggcgaagcg gacgctgtta agctggaagt
     1921 gcagtacatg tacgaggcgc tgatgtgcgg tctgctgccg gctgttgagg aataccacaa
     1981 caaagacgat ttcggtccgt gcaagacccc aggtagcccg gccaccgact tcggcaccct
     2041 gcgtgcggtt gctctggtga agaaaagcaa caaagacatc aactggaaca acatcaaggg
     2101 caagaagagc tgccacaccg gtgtgggcga catcgctggc tgggttatcc cggtgagcct
     2161 gatccgtcgt cagaacgata acagcgacat cgatagcttc ttcggcgaga gctgcgcgcc
     2221 gggcagcgac accaaaagca acctgtgcaa gctgtgcatc ggtgacccga agaacagcgc
     2281 ggctaacacc aaatgcagcc tgagcgacaa ggaagcgtac tacggtaacc agggcgcttt
     2341 ccgttgcctg gtggagaaag gtgacgttgc tttcgtgccg cacaccgtgg ttttcgaaaa
     2401 caccgatggc aagaacccgg cggtttgggc taagaacctg aaaagcgaag acttcgagct
     2461 gctgtgcctg gatggtagcc gtgcgccggt gagcaactac aagagctgca aactgagcgg
     2521 tatcccgccg ccggctatcg ttacccgtga ggaaagcatc agcgacgtgg ttcgtatcgt
     2581 ggctaaccag cagagcctgt acggtcgtaa aggcttcgag aaggatatgt tccagctgtt
     2641 cagcagcaac aagggtaaca acctgctgtt caacgacaac acccagtgcc tggccacctt
     2701 cgatcgtcag ccgaaagaca tcatggaaga ttacttcggc aagccgtact acaccaccgt
     2761 gtacggcgcg tcccgctccg ctatgtcctc cgaactgatt tccgcctgta ccattaaaca
     2821 ctgctcgaat tctttggaag ttttgtttca aggtccaact gctgccgccg ctgtgagcaa
     2881 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2941 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3001 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3061 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3121 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3181 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3241 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3301 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3361 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3421 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3481 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3541 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg gaggtggtca
     3601 ccatcaccat caccaccatc accatcacta atagaagctt gtcgagaagt actagaggat
     3661 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     3721 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     3781 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3841 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg
     3901 atcactgctt gagcctagga gatccgaacc agataagtga aatctagttc caaactattt
     3961 tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat ctattttgtc
     4021 actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc ccaactattt
     4081 tgtccgccca cagcggggca tttttcttcc tgttatgttt ttaatcaaac atcctgccaa
     4141 ctccatgtga caaaccgtca tcttcggcta ctttttctct gtcacagaat gaaaattttt
     4201 ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat atcaacgctt atttgcagcc
     4261 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4321 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4381 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4441 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4501 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4561 cgttcttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4621 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4681 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     4741 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4801 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4861 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4921 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4981 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5041 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5101 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5161 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5221 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5281 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5341 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5401 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5461 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5521 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5581 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5641 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5701 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5761 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6001 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6541 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6601 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6661 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagac
     6721 cagccgcgta acctggcaaa atcggttacg gttgagtaat aaatggatgc cctgcgtaag
     6781 cgggtgtggg cggacaataa agtcttaaac tgaacaaaat agatctaaac tatgacaata
     6841 aagtcttaaa ctagacagaa tagttgtaaa ctgaaatcag tccagttatg ctgtgaaaaa
     6901 gcatactgga cttttgttat ggctaaagca aactcttcat tttctgaagt gcaaattgcc
     6961 cgtcgtatta aagaggggcg tggccaaggg catggtaaag actatattcg cggcgttgtg
     7021 acaatttacc gaacaactcc gcggccggga agccgatctc ggcttgaacg aattgttagg
     7081 tggcggtact tgggtcgata tcaaagtgca tcacttcttc ccgtatgccc aactttgtat
     7141 agagagccac tgcgggatcg tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa
     7201 gcgcgttggc ctcatgcttg aggagattga tgagcgcggt ggcaatgccc tgcctccggt
     7261 gctcgccgga gactgcgaga tcatagatat agatctcact acgcggctgc tcaaacttgg
     7321 gcagaacgta agccgcgaga gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga
     7381 tgaatgtctt actacggagc aagttcccga ggtaatcgga gtccggctga tgttgggagt
     7441 aggtggctac gtctccgaac tcacgaccga aaagatcaag agcagcccgc atggatttga
     7501 cttggtcagg gccgagccta catgtgcgaa tgatgcccat acttgagcca cctaactttg
     7561 ttttagggcg actgccctgc tgcgtaacat cgttgctgct ccataacatc aaacatcgac
     7621 ccacggcgta acgcgcttgc tgcttggatg cccgaggcat agactgtaca aaaaaacagt
     7681 cataacaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc
     7741 tggaccagtt gcgtgagcgc atacgctact tgcattacag tttacgaacc gaacaggctt
     7801 atgtcaactg ggttcgtgcc ttcatccgtt tccacggtgt gcgtcacccg gcaaccttgg
     7861 gcagcagcga agtcgaggca tttctgtcct ggctggcgaa cgagcgcaag gtttcggtct
     7921 ccacgcatcg tcaggcattg gcggccttgc tgttcttcta cggcaaggtg ctgtgcacgg
     7981 atctgccctg gcttcaggag atc
//
