LOCUS       sequence-343065-        7102 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses GFP-endoNA2 fusion protein for detection of polysialic 
            acid. The endoNA2 is a catalytically inactive form of endosialidase 
            from bacteriophage PK1A2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  AUTHORS   Jokilammi A, Ollikka P, Korja M, Jakobsson E, Loimaranta V, Haataja 
            S, Hirvonen H, Finne J
  TITLE     Construction of antibody mimics from a noncatalytic enzyme-detection
            of polysialic acid.
  JOURNAL   J Immunol Methods. 2004 Dec;295(1-2):149-60. doi: 
            10.1016/j.jim.2004.10.006. Epub 2004 Nov 14.
  PUBMED    15627620
REFERENCE   2  (bases 1 to 7102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Immunol 
            Methods."; date: "2004-12"; volume: "295(1-2):149-60. doi"; pages: "
            10.1016/j.jim.2004.10.006. Epub 2004 Nov 14"
COMMENT     Sequence Label: pFEndoNA2
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..52
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(76..90,91..96,97..113,114..120)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    96..112
                     /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)."
     protein_bind    128..144
                     /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             163..174
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             193..210
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      229..238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(235..237,238..240,241..948)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(235..237,238..240,241..948)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(280..301)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(541..560)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     888..909
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      3915..4009
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4053..4712
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4119..4138)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4777..4863
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4910..4932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5018..5158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5073..5092
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6103..6894,6895..6963))
                     /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     6726..6745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6964..7068)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
       61 cttcacctcg agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata
      121 gattcaattg tgagcggata acaatttcac acagaattca ttaaagagga gaaattaact
      181 atgagaggat ctcaccatca ccatcaccat gatccccggg taccggtcgc caccatggtg
      241 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      301 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      361 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      421 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      481 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      541 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      601 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      661 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      721 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      781 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      841 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      901 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacgg gatcgcaaat
      961 aactatactg atagtaaaag tgctgaaaca ttggaaaatg cgcgtaaaga ggcttttgct
     1021 cagtctaaag atgttttgaa tatggcaaaa gcacactcaa atagtgttgc tagaacaact
     1081 ttagaaactg ctgaagaaca tgcaaatagt gttgccagaa caactttaga aactgctgaa
     1141 gaacatgcaa ataaaaaatc agctgaggcg ttagcaagcg ctaatgtgta tgcagacagc
     1201 aagtcttctc acacactaaa aactgcaaat agctataccg atgtgactgt aagtaattcg
     1261 actaagaaag caatccgtga atcgaatcaa tacacagatc ataaattccg tcaacttgac
     1321 aaccggttag ataagctgta cacggatcaa ttcaagagta aaatagcagg tgcaatctgg
     1381 cgtaacttgg atgacaagct caccgaggtt gtatcgctta aagattttgg agccaaaggt
     1441 gatggtaaga caaacgacca agatgcagta aatgcagcga tggcttcagg taagagaatt
     1501 gacggtgctg gtgctactta caaagtatca tctttacctg atatggagcg attctataac
     1561 acccgcttcg tatgggaacg tttatcaggt caacctcttt actatgtgag taaaggtttt
     1621 atcaatggtg aactctataa aatcacggat aacccttatt acaatgcttg gcctcaagac
     1681 aaagcgtttg tatatgagaa cgtgatatat gcaccttaca tgggtagcga ccgtcatggt
     1741 gttagtcgtc tgcatgtatc atgggttaag tctggtgacg atggtcaaac atggtctaca
     1801 ccagagtggt taactgatct gcatccagat taccctacag tgaactatca ttgtatgagc
     1861 atgggtgtat gtcgcaaccg tctgtttgcc atgattgaaa cacgtacttt agccaagaac
     1921 gcactaacca attgtgcatt gtgggatcgc cctatgtctc gtagtctgca tcttacgggt
     1981 ggtatcacta aggctgcaaa tcagcgatat gcaacaatac atgtaccaga tcacggacta
     2041 ttcgtgggcg attttgttaa cttctctaat tctgcggtaa caggtgtatc aggtgatatg
     2101 actgttgcaa cggtaataga taaggacaac ttcacggttc ttacacctaa ccagcagact
     2161 tcagatttga ataacgctgg aaagaattgg cacatgggta cttctttcca taagtctccg
     2221 tggcgtaaga cagatcttgg tctaatccct catgtcacag aggtgcatag ctttgctact
     2281 attgataaca atggctttgt tatgggctat catcaaggtg atgtagctcc acgagaagtt
     2341 gggcttttct acttccctga tgctttcaat agcccatcta attatgttcg tcgtcagata
     2401 ccatctgagt atgaaccaga tgcggcagag ccatgcatca agtactatga cggtgtatta
     2461 taccttatca ctcgtggtac tcgtggcgac cgactaggaa gctctctgca tcgtagtaga
     2521 gatataggtc agacttggga gtcactaaga tttccacata atgtgtatca tactacttta
     2581 ccgtttgcta aggtaggaga tgaccttatt atgtttggtt cagaacgtgc agaaaatgaa
     2641 tgggaagcag gtgcaccaga tgatcgttac aaggcatctt atcctcgtac cttctatgca
     2701 cgattgaatg taaacaattg gaatgcagct gatattgaat gggttaacat cacagaccaa
     2761 atctatcagg gtgacattgt ggactctagt gtaggtgtag gttcggtagt agttaaagac
     2821 agctacattt actatatctt tggtggcgaa aaccatttca acccaatgac ttatggtgac
     2881 aacagagata aagacccatt taaaggtcat ggacacccta ctgatatata ctgctataag
     2941 atgcagattg caaatgacag tcgtgtatct cgtaagttta catatggtgc aactcccgga
     3001 caggctatac ctaccttcat gggtactgat ggtattcgta ctattcctgc tccacttcac
     3061 ttatcaggag aggtagttgc cgcagatatg actgtcgaac atcttaccct gaaggcaagt
     3121 acaagtgcta atattcgctc agagatgctg atggaaggtg aatatgggtt cattggtaag
     3181 actataccaa aggataaccc aactgcccaa cgtgtcatca tctctggtgg agggggtaca
     3241 gcagcagata ccggtgcgca aattacctta catggagcag gttctagtac ttctagacgc
     3301 gcagtatata atgccaatga gcacttgttc caaagcgggg ctatcatgcc ctacaatgat
     3361 aacgtgtatc ctgctggcgg ccctcgtaat agattcacta ctatccatct tacaagtgac
     3421 cctattatta cgtctgatgc cactcataag tatggatcac aaggtattga tgagagtgta
     3481 ctaaaggcat ggggtaaggt tagcttcaag cagtacaagt tgattggtga gatgtctcgt
     3541 ggtgtgcacc acactcactt tggtgtattg gcacaagaca ttgtagctgc ttttgcatcc
     3601 gaagggttag atgcaatcga cttcggcatt gtctccttcg aagaaggtca gtttggtgtt
     3661 agatatagtg aaatcttaat cttggaggct gcttatactc gttatcgttt agataagtta
     3721 gaggagatgt atgccactaa taaaatcagt taagcaagct gctgtactcc agaatacaga
     3781 agaacttatt caatcaggac gtgaccctaa gcaggcatac gccattgcca aggatgtaca
     3841 agtaaagcgg ccgatccgca tgcgagctcg gtaccccggg tcgacctgca gccaagctta
     3901 attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     3961 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagctagct
     4021 tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     4081 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     4141 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     4201 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     4261 atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     4321 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     4381 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     4441 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     4501 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     4561 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     4621 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     4681 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     4741 aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg
     4801 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     4861 aatccgccct ctagagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     5041 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     5101 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6241 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7081 aaaagtgcca cctgacgtct aa
//
