LOCUS       Exported                6320 bp ds-DNA     circular SYN 17-APR-2020
DEFINITION  pcDNA3.4_mIgG2-mMxr8a plasmid was digested with EcoRI-HF and AgeI 
            restriction enzymes to remove the mIgG2-mxr8 cassette. Remaining 
            backbone of pcDNA3.1 was used to clone CEACAM6-His amplicon 
            amplified from RC202454 plasmid using 030218.1 and 030218.3 primer 
            set. Gibson assembly reagent was used for cloning.  Clones were PCR 
            confirmed for CEACAM6-His insertion, plasmid was isolated and 
            confirmed by sequencing. This CEACAM6(3D)-His has a point mutation 
            (GGA to GCA) to replace  'G-glycine' with 'A-alanine' at the site of
            lipidation required for GPI anchoring.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  AUTHORS   Zhang R, Kim AS, Fox JM, Nair S, Basore K, Klimstra WB, Rimkunas R, 
            Fong RH, Lin H, Poddar S, Crowe JE, Doranz BJ, Fremont DH, Diamond 
            MS.
  TITLE     Mxra8 is a receptor for multiple arthritogenic alphaviruses.
  JOURNAL   Nature
  PUBMED    29769725
REFERENCE   2  (bases 1 to 6320)
  AUTHORS   fleckenstein lab
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Apr 17, 2020 from SnapGene 5.1.0
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-018-0121-3"; journalName: "Nature"; volume: "557"; 
            pages: "570-574"
COMMENT     original pCDNA3.4_mIgG2_mMxr8a plasmid was obtained from the 
            laboratory of Dr. Michael Diamond at Washington University.
FEATURES             Location/Qualifiers
     source          1..6320
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             938..997
                     /codon_start=1
                     /product="human interleukin-2 signal sequence"
                     /label=IL-2 signal sequence
                     /translation="MYRMQLLSCIALSLALVTNS"
     primer_bind     972..1024
                     /label=030218.1
                     /note="_CEACAM6-RC202454_3D_pFuse_F (pCDNA3.4)"
     CDS             1004..1861
                     /codon_start=1
                     /product="3 domains of CEACAM6 with point mutation to 
                     abolish lipidation site for GPI anchoring"
                     /label=CEACAM6_3domains
                     /translation="KLTIESTPFNVAEGKEVLLLAHNLPQNRIGYSWYKGERVDGNSLI
                     VGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQF
                     HVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQLSNGN
                     MTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYRPGENLN
                     LSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQAHNSATGLNRTTVTM
                     ITVSA"
     primer_bind     complement(1840..1889)
                     /label=030218.3
                     /note="_CEACAM6-RC202454_His_pFuse_R (pCDNA3.4)"
     misc_feature    1860
                     /label=point mutation to replace lipidation site
                     /note="G-glycine codon is changed with A-alanine codon to 
                     replace the lipidation site require GPI-anchoring.   "
     CDS             1868..1885
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1914..2138
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2184..2612
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2626..2956
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2807..2942
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3023..3817
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3993..4114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4163..4179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4187..4203
                     /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)."
     promoter        complement(4211..4241)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4256..4277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4565..5153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5324..6184)
                     /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"
     promoter        complement(6185..6289)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtcgactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
      841 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagtt
      901 aagcttggta ccgagctcgg atccagtacc cttcaccatg tacaggatgc aactcctgtc
      961 ttgcattgca ctaagtcttg cacttgtcac gaattcgata tcgaagctca ctattgaatc
     1021 cacgccattc aatgtcgcag aggggaagga ggttcttcta ctcgcccaca acctgcccca
     1081 gaatcgtatt ggttacagct ggtacaaagg cgaaagagtg gatggcaaca gtctaattgt
     1141 aggatatgta ataggaactc aacaagctac cccagggccc gcatacagtg gtcgagagac
     1201 aatatacccc aatgcatccc tgctgatcca gaacgtcacc cagaatgaca caggattcta
     1261 taccctacaa gtcataaagt cagatcttgt gaatgaagaa gcaaccggac agttccatgt
     1321 atacccggag ctgcccaagc cctccatctc cagcaacaac tccaaccccg tggaggacaa
     1381 ggatgctgtg gccttcacct gtgaacctga ggttcagaac acaacctacc tgtggtgggt
     1441 aaatggtcag agcctcccgg tcagtcccag gctgcagctg tccaatggca acatgaccct
     1501 cactctactc agcgtcaaaa ggaacgatgc aggatcctat gaatgtgaaa tacagaaccc
     1561 agcgagtgcc aaccgcagtg acccagtcac cctgaatgtc ctctatggcc cagatgtccc
     1621 caccatttcc ccctcaaagg ccaattaccg tccaggggaa aatctgaacc tctcctgcca
     1681 cgcagcctct aacccacctg cacagtactc ttggtttatc aatgggacgt tccagcaatc
     1741 cacacaagag ctctttatcc ccaacatcac tgtgaataat agcggatcct atatgtgcca
     1801 agcccataac tcagccactg gcctcaatag gaccacagtc acgatgatca cagtctctgc
     1861 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     1921 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1981 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2041 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2101 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2161 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2221 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2281 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2341 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2401 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2461 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2521 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     2581 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2641 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     2701 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2761 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2821 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2881 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2941 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3001 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3061 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3121 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3181 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3241 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3301 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3361 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3421 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3481 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3541 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3601 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3661 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3721 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3781 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgcgaaa
     3841 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3901 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3961 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4021 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4081 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4141 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4201 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4261 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4321 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4381 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4441 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4501 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4561 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4621 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4681 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4741 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
     4801 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4861 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4921 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4981 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5041 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5101 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5161 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5221 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5281 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5341 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5401 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5461 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5521 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5581 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5641 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5701 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5761 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5821 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5881 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5941 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6001 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6061 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6121 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6181 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6241 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6301 gaaaagtgcc acctgacgtc
//
