LOCUS       sequence-390589-        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 E540D
FEATURES             Location/Qualifiers
     source          1..8003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        205..296
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     221..238
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2883..2906
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2922..2924,2925..3635)
                     /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(2964..2985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3225..3244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3572..3593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3648..3677
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     polyA_signal    3813..3947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3850..3869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3904..3923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3976..4141
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4325..4779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4412..4431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4621..4642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4805..4909
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4910..4978,4979..5770)
                     /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(5128..5147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5941..6529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6430..6449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6683..6700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6834..7058
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(7125..7658)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7828..7833,7834..7850,7851..7856))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 cttctacggc aaggtgctgt gcacggatct gccctggctt caggagatcg gaagacctcg
       61 gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa gtggttcgca tcctcggttt
      121 tctggaaggc gagcatcgtt tgttcgccca ggactctagc tatagttcta gtggttggct
      181 acgtatactc cggaatatta atagatcatg gagataatta aaatgataac catctcgcaa
      241 ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct ataaatattc
      301 cggattattc ataccgtccc accatcgggc gcggatccat ggctcctacc ttccagaccg
      361 ctctgttctt caccatcatc tccctgtcct tcgctatggc cccgaacgca aagcaagtcc
      421 gctggtgtgc tatcagcgac ctggagcaga agaaatgcaa cgatctggtt ggtagctgca
      481 acgtgccgga catcaccctg gtgtgcgttc tgcgtagcag caccgaagat tgcatgaccg
      541 cgatcaagga cggtcaggcg gatgctatgt tcctggacag cggcgaggtg tacgaagcga
      601 gcaaagatcc gtacaacctg aagccgatca tcgctgaacc gtacagcagc aaccgtgacc
      661 tgcagaagtg cctgaaagag cgtcagcagg cgctggctaa gaaaatgatc ggtcactaca
      721 tcccgcagtg cgatgagaaa ggcaactacc aaccgcagca gtgccacggt agcaccggtc
      781 actgctggtg cgttaacgcg atgggtgaaa agatcagcgg taccaacacc ccgccgggtc
      841 agacccgtgc tacctgcgag cgtcacgaac tgccgaagtg cctgaaagaa cgtcaggtgg
      901 cgctgggtgg cgacgagaaa gttctgggtc gtttcgtgcc gcagtgcgat gaaaagggca
      961 actacgagcc gcagcagttc cacggtagca ccggctactc ctggtgtgtt aacgcgatcg
     1021 gcgaggaaat cgctggcacc aaaaccccgc cgggcaagat cccggctacc tgccagaagc
     1081 acgacctggt gaccacctgc cactacgcgg ttgctatggt gaagaaaagc agcgcgttcc
     1141 agttcaacca gctgaagggc aaacgtagct gccacagcgg tgttagcaaa accgacggct
     1201 ggaaggcgct ggtgaccgtt ctggtggaaa agaaactgct gagctgggat ggtccggcga
     1261 aagagagcat ccagcgtgct atgagcaagt tcttcagcgt gagctgcatt ccgggtgcta
     1321 cccagaccaa cctgtgcaag cagtgcaaag gcgaggaagg caagaactgc aaaaacagcc
     1381 acgacgaacc gtactacggt aactacggcg cgttccgttg cctgaaggaa gacatgggtg
     1441 acgttgcgtt cctgcgtagc accgctctga gcgacgagca cagcgaagtg tacgagctgc
     1501 tgtgcccgga taacacccgt aagccgctga acaagtacaa agagtgcaac ctgggtaccg
     1561 ttccggctgg caccgtggtt acccgtaaga tcagcgacaa aaccgaagac atcaacaact
     1621 tcctgatgga ggcgcagaag cgtcagtgca aactgttcag cagcgctcac ggcaaggacc
     1681 tgatgttcga cgatagcacc ctgcagctgg ctctgctgag cagcgaggtt gatgctttcc
     1741 tgtacctggg tgtgaaactg ttccacgcga tgaaggctct gaccggcgac gctcacctgc
     1801 cgagcaagaa caaagtgcgt tggtgcacca tcaacaagct ggaaaagatg aaatgcgacg
     1861 attggagcgc ggttagcggt ggcgcgatcg cttgcaccga ggctagctgc ccgaagggct
     1921 gcgtgaaaca gatcctgaag ggcgaagcgg acgctgttaa gctggaagtg cagtacatgt
     1981 acgaggcgct gatgtgcggt ctgctgccgg ctgttgatga ataccacaac aaagacgatt
     2041 tcggtccgtg caagacccca ggtagcccgt acaccgactt cggcaccctg cgtgcggttg
     2101 ctctggtgaa gaaaagcaac aaagacatca actggaacaa catcaagggc aagaagagct
     2161 gccacaccgg tgtgggcgac atcgctggct gggttatccc ggtgagcctg atccgtcgtc
     2221 agaacgataa cagcgacatc gatagcttct tcggcgagag ctgcgcgccg ggcagcgaca
     2281 ccaaaagcaa cctgtgcaag ctgtgcatcg gtgacccgaa gaacagcgcg gctaacacca
     2341 aatgcagcct gagcgacaag gaagcgtact acggtaacca gggcgctttc cgttgcctgg
     2401 tggagaaagg tgacgttgct ttcgtgccgc acaccgtggt tttcgaaaac accgatggca
     2461 agaacccggc ggtttgggct aagaacctga aaagcgaaga cttcgagctg ctgtgcctgg
     2521 atggtagccg tgcgccggtg agcaactaca agagctgcaa actgagcggt atcccgccgc
     2581 cggctatcgt tacccgtgag gaaagcatca gcgacgtggt tcgtatcgtg gctaaccagc
     2641 agagcctgta cggtcgtaaa ggcttcgaga aggatatgtt ccagctgttc agcagcaaca
     2701 agggtaacaa cctgctgttc aacgacaaca cccagtgcct gatcaccttc gatcgtcagc
     2761 cgaaagacat catggaagat tacttcggca agccgtacta caccaccgtg tacggcgcgt
     2821 cccgctccgc tatgtcctcc gaactgattt ccgcctgtac cattaaacac tgctcgaatt
     2881 ctttggaagt tttgtttcaa ggtccaactg ctgccgccgc tgtgagcaag ggcgaggagc
     2941 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3001 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3061 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     3121 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3181 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3241 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3301 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     3361 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     3421 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3481 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     3541 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3601 ccgggatcac tctcggcatg gacgagctgt acaagtccgg aggtggtcac catcaccatc
     3661 accaccatca ccatcactaa tagaagcttg tcgagaagta ctagaggatc ataatcagcc
     3721 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3781 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3841 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3901 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga tcactgcttg
     3961 agcctaggag atccgaacca gataagtgaa atctagttcc aaactatttt gtcattttta
     4021 attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca ctcttcccta
     4081 aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt gtccgcccac
     4141 agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac tccatgtgac
     4201 aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc tgtcatctct
     4261 tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct gaatggcgaa
     4321 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4381 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4441 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4501 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4561 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttcttaata
     4621 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4681 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4741 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     4801 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4861 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4921 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4981 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5041 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5101 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5161 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5221 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5281 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5341 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5401 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5461 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5521 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5581 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5641 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5701 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5761 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5821 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5881 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5941 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6001 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6061 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6121 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6181 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6241 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6301 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6361 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6421 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6481 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6541 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6601 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     6661 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     6721 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagacc agccgcgtaa
     6781 cctggcaaaa tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc gggtgtgggc
     6841 ggacaataaa gtcttaaact gaacaaaata gatctaaact atgacaataa agtcttaaac
     6901 tagacagaat agttgtaaac tgaaatcagt ccagttatgc tgtgaaaaag catactggac
     6961 ttttgttatg gctaaagcaa actcttcatt ttctgaagtg caaattgccc gtcgtattaa
     7021 agaggggcgt ggccaagggc atggtaaaga ctatattcgc ggcgttgtga caatttaccg
     7081 aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt
     7141 gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact
     7201 gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc
     7261 tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctcgccggag
     7321 actgcgagat catagatata gatctcacta cgcggctgct caaacttggg cagaacgtaa
     7381 gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta
     7441 ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta ggtggctacg
     7501 tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg
     7561 ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga
     7621 ctgccctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc cacggcgtaa
     7681 cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc ataacaagcc
     7741 atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct ggaccagttg
     7801 cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta tgtcaactgg
     7861 gttcgtgcct tcatccgttt ccacggtgtg cgtcacccgg caaccttggg cagcagcgaa
     7921 gtcgaggcat ttctgtcctg gctggcgaac gagcgcaagg tttcggtctc cacgcatcgt
     7981 caggcattgg cggccttgct gtt
//
