LOCUS       sequence-390588-        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 E540A
FEATURES             Location/Qualifiers
     source          1..8003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        176..267
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     192..209
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2854..2877
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2893..2895,2896..3606)
                     /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(2935..2956)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3196..3215)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3543..3564
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3619..3648
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    3784..3918
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3821..3840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3875..3894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3947..4112
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4296..4750
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4383..4402)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4592..4613
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4776..4880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4881..4949,4950..5741)
                     /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(5099..5118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5912..6500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6401..6420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6654..6671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6805..7029
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(7096..7629)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7799..7804,7805..7821,7822..7827))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 tgccctggct tcaggagatc ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga
       61 ccccggatga agtggttcgc atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc
      121 aggactctag ctatagttct agtggttggc tacgtatact ccggaatatt aatagatcat
      181 ggagataatt aaaatgataa ccatctcgca aataaataag tattttactg ttttcgtaac
      241 agttttgtaa taaaaaaacc tataaatatt ccggattatt cataccgtcc caccatcggg
      301 cgcggatcca tggctcctac cttccagacc gctctgttct tcaccatcat ctccctgtcc
      361 ttcgctatgg ccccgaacgc aaagcaagtc cgctggtgtg ctatcagcga cctggagcag
      421 aagaaatgca acgatctggt tggtagctgc aacgtgccgg acatcaccct ggtgtgcgtt
      481 ctgcgtagca gcaccgaaga ttgcatgacc gcgatcaagg acggtcaggc ggatgctatg
      541 ttcctggaca gcggcgaggt gtacgaagcg agcaaagatc cgtacaacct gaagccgatc
      601 atcgctgaac cgtacagcag caaccgtgac ctgcagaagt gcctgaaaga gcgtcagcag
      661 gcgctggcta agaaaatgat cggtcactac atcccgcagt gcgatgagaa aggcaactac
      721 caaccgcagc agtgccacgg tagcaccggt cactgctggt gcgttaacgc gatgggtgaa
      781 aagatcagcg gtaccaacac cccgccgggt cagacccgtg ctacctgcga gcgtcacgaa
      841 ctgccgaagt gcctgaaaga acgtcaggtg gcgctgggtg gcgacgagaa agttctgggt
      901 cgtttcgtgc cgcagtgcga tgaaaagggc aactacgagc cgcagcagtt ccacggtagc
      961 accggctact cctggtgtgt taacgcgatc ggcgaggaaa tcgctggcac caaaaccccg
     1021 ccgggcaaga tcccggctac ctgccagaag cacgacctgg tgaccacctg ccactacgcg
     1081 gttgctatgg tgaagaaaag cagcgcgttc cagttcaacc agctgaaggg caaacgtagc
     1141 tgccacagcg gtgttagcaa aaccgacggc tggaaggcgc tggtgaccgt tctggtggaa
     1201 aagaaactgc tgagctggga tggtccggcg aaagagagca tccagcgtgc tatgagcaag
     1261 ttcttcagcg tgagctgcat tccgggtgct acccagacca acctgtgcaa gcagtgcaaa
     1321 ggcgaggaag gcaagaactg caaaaacagc cacgacgaac cgtactacgg taactacggc
     1381 gcgttccgtt gcctgaagga agacatgggt gacgttgcgt tcctgcgtag caccgctctg
     1441 agcgacgagc acagcgaagt gtacgagctg ctgtgcccgg ataacacccg taagccgctg
     1501 aacaagtaca aagagtgcaa cctgggtacc gttccggctg gcaccgtggt tacccgtaag
     1561 atcagcgaca aaaccgaaga catcaacaac ttcctgatgg aggcgcagaa gcgtcagtgc
     1621 aaactgttca gcagcgctca cggcaaggac ctgatgttcg acgatagcac cctgcagctg
     1681 gctctgctga gcagcgaggt tgatgctttc ctgtacctgg gtgtgaaact gttccacgcg
     1741 atgaaggctc tgaccggcga cgctcacctg ccgagcaaga acaaagtgcg ttggtgcacc
     1801 atcaacaagc tggaaaagat gaaatgcgac gattggagcg cggttagcgg tggcgcgatc
     1861 gcttgcaccg aggctagctg cccgaagggc tgcgtgaaac agatcctgaa gggcgaagcg
     1921 gacgctgtta agctggaagt gcagtacatg tacgaggcgc tgatgtgcgg tctgctgccg
     1981 gctgttgcgg aataccacaa caaagacgat ttcggtccgt gcaagacccc aggtagcccg
     2041 tacaccgact tcggcaccct gcgtgcggtt gctctggtga agaaaagcaa caaagacatc
     2101 aactggaaca acatcaaggg caagaagagc tgccacaccg gtgtgggcga catcgctggc
     2161 tgggttatcc cggtgagcct gatccgtcgt cagaacgata acagcgacat cgatagcttc
     2221 ttcggcgaga gctgcgcgcc gggcagcgac accaaaagca acctgtgcaa gctgtgcatc
     2281 ggtgacccga agaacagcgc ggctaacacc aaatgcagcc tgagcgacaa ggaagcgtac
     2341 tacggtaacc agggcgcttt ccgttgcctg gtggagaaag gtgacgttgc tttcgtgccg
     2401 cacaccgtgg ttttcgaaaa caccgatggc aagaacccgg cggtttgggc taagaacctg
     2461 aaaagcgaag acttcgagct gctgtgcctg gatggtagcc gtgcgccggt gagcaactac
     2521 aagagctgca aactgagcgg tatcccgccg ccggctatcg ttacccgtga ggaaagcatc
     2581 agcgacgtgg ttcgtatcgt ggctaaccag cagagcctgt acggtcgtaa aggcttcgag
     2641 aaggatatgt tccagctgtt cagcagcaac aagggtaaca acctgctgtt caacgacaac
     2701 acccagtgcc tgatcacctt cgatcgtcag ccgaaagaca tcatggaaga ttacttcggc
     2761 aagccgtact acaccaccgt gtacggcgcg tcccgctccg ctatgtcctc cgaactgatt
     2821 tccgcctgta ccattaaaca ctgctcgaat tctttggaag ttttgtttca aggtccaact
     2881 gctgccgccg ctgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2941 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     3001 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     3061 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     3121 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     3181 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     3241 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     3301 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     3361 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     3421 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     3481 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     3541 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     3601 tacaagtccg gaggtggtca ccatcaccat caccaccatc accatcacta atagaagctt
     3661 gtcgagaagt actagaggat cataatcagc cataccacat ttgtagaggt tttacttgct
     3721 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3781 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3901 tcttatcatg tctggatctg atcactgctt gagcctagga gatccgaacc agataagtga
     3961 aatctagttc caaactattt tgtcattttt aattttcgta ttagcttacg acgctacacc
     4021 cagttcccat ctattttgtc actcttccct aaataatcct taaaaactcc atttccaccc
     4081 ctcccagttc ccaactattt tgtccgccca cagcggggca tttttcttcc tgttatgttt
     4141 ttaatcaaac atcctgccaa ctccatgtga caaaccgtca tcttcggcta ctttttctct
     4201 gtcacagaat gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat
     4261 atcaacgctt atttgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     4321 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4381 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4441 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4501 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4561 ccctttgacg ttggagtcca cgttcttaat agtggactct tgttccaaac tggaacaaca
     4621 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4681 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4741 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4801 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4861 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4921 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4981 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5041 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5101 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5161 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5221 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5281 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5341 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5401 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5461 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5521 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5581 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5641 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5701 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5761 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5821 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5881 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6001 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6061 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6121 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6181 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6241 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6301 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6361 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6421 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6481 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6541 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6601 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6661 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     6721 gtatttcaca ccgcatagac cagccgcgta acctggcaaa atcggttacg gttgagtaat
     6781 aaatggatgc cctgcgtaag cgggtgtggg cggacaataa agtcttaaac tgaacaaaat
     6841 agatctaaac tatgacaata aagtcttaaa ctagacagaa tagttgtaaa ctgaaatcag
     6901 tccagttatg ctgtgaaaaa gcatactgga cttttgttat ggctaaagca aactcttcat
     6961 tttctgaagt gcaaattgcc cgtcgtatta aagaggggcg tggccaaggg catggtaaag
     7021 actatattcg cggcgttgtg acaatttacc gaacaactcc gcggccggga agccgatctc
     7081 ggcttgaacg aattgttagg tggcggtact tgggtcgata tcaaagtgca tcacttcttc
     7141 ccgtatgccc aactttgtat agagagccac tgcgggatcg tcaccgtaat ctgcttgcac
     7201 gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg aggagattga tgagcgcggt
     7261 ggcaatgccc tgcctccggt gctcgccgga gactgcgaga tcatagatat agatctcact
     7321 acgcggctgc tcaaacttgg gcagaacgta agccgcgaga gcgccaacaa ccgcttcttg
     7381 gtcgaaggca gcaagcgcga tgaatgtctt actacggagc aagttcccga ggtaatcgga
     7441 gtccggctga tgttgggagt aggtggctac gtctccgaac tcacgaccga aaagatcaag
     7501 agcagcccgc atggatttga cttggtcagg gccgagccta catgtgcgaa tgatgcccat
     7561 acttgagcca cctaactttg ttttagggcg actgccctgc tgcgtaacat cgttgctgct
     7621 ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat
     7681 agactgtaca aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc
     7741 gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag
     7801 tttacgaacc gaacaggctt atgtcaactg ggttcgtgcc ttcatccgtt tccacggtgt
     7861 gcgtcacccg gcaaccttgg gcagcagcga agtcgaggca tttctgtcct ggctggcgaa
     7921 cgagcgcaag gtttcggtct ccacgcatcg tcaggcattg gcggccttgc tgttcttcta
     7981 cggcaaggtg ctgtgcacgg atc
//
