LOCUS       sequence-393687-        7249 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of secreted mouse CES2A S227A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7249)
  AUTHORS   Wei W, Riley NM, Lyu X, Shen X, Guo J, Raun SH, Zhao M, Moya-Garzon 
            MD, Basu H, Sheng-Hwa Tung A, Li VL, Huang W, Wiggenhorn AL, 
            Svensson KJ, Snyder MP, Bertozzi CR, Long JZ
  TITLE     Organism-wide, cell-type-specific secretome mapping of exercise 
            training in mice.
  JOURNAL   Cell Metab. 2023 Apr 28:S1550-4131(23)00138-9. doi: 
            10.1016/j.cmet.2023.04.011.
  PUBMED    37141889
REFERENCE   2  (bases 1 to 7249)
  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: "Cell Metab.
            2023 Apr 28:S1550-4131(23)00138-9. doi: 10.1016/j.cmet.2023.04.011."
COMMENT     Sequence Label: pDEST40-CES2A-Delta-C-S227A
FEATURES             Location/Qualifiers
     source          1..7249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1782..1985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2030..2049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2030..2048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2078..2102
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2207..2230
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3824..3841
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3864..3888)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3914..3955
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3965..3982
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4005..4022)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4011..4235
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4281..4709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4368..4387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4578..4599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4718..4738)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4723..5052
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4903..5038
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4965..4984
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5119..5913
                     /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"
     primer_bind     complement(5173..5192)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5783..5802
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6089..6210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6126..6145)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6180..6199
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6259..6275)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6259..6275)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6272..6294)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6283..6299
                     /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(join(6307..6313,6314..6331,6332..6337))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6352..6373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6490..6507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6661..7249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6741..6760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2041 actataggga gacccaagct ggctagttaa gctatcaaca agtttgtaca aaaaagcagg
     2101 ctccgcggcc gcccccttca ccgccaccat gccattggct agacttcctg gttggctgtg
     2161 tgttgttgcc tgtgggctcc tgcttctcct ccagcatgtg catggcgact acaaggatga
     2221 cgacgataag gggggcggtc aggactcagc cagccccatc aggaacaccc acaggggtca
     2281 ggtgagaggc agctttgtcc acgtgaagga cactaaatct ggtgtccatg ccttcctggg
     2341 aatccccttt gccaagcctc ctgtaggact actgcgcttt gcacctcctg aggaccctga
     2401 gccatggagt ggtgtgagag atgggacctc acagccggcc atgtgtctgc agcctgatat
     2461 aatgaattta gaggatgcga aggaaatgaa tctgatcctg cctcccatct ctatgtccga
     2521 ggactgcctg tatctcaaca tctacacacc aacccacgcc caggagggct ctaacctgcc
     2581 tgtgatggta tggatccacg gtggtggact ggttgtagga tcagcttcca tgaatgatgt
     2641 atctaaactg gcagccactg aggaaatagt aattgtcgcc atccagtatc gcttgggggt
     2701 ccttggcttc ttcagcactg gagaccagca tgccagaggc aactggggat acctggacca
     2761 agtggctgcc ttacgctggg tgcagaagaa tattgcttac tttggaggca accgtgacag
     2821 ggtcactata tttggagtcg cagcaggagg tacaagtgtg tcttctcata ttctgtcccc
     2881 aatgtccaaa ggactctttc atggagccat tatgcagagt ggagtggccc tgctgcctga
     2941 cctcatctct gacacttctg aggtggtcta caagacagta gccaacctat ctggttgtga
     3001 agccacagac tcagaagccc tgatccactg cctgagagca aagagtaaac aagagatcct
     3061 ggctattaac caggtcttca agatgatccc tgctgtggtg gatggggagt tcctacccaa
     3121 gcatcctcag gagctgctga cctctatgga ttttcaccct gtccccagca tcattggtgt
     3181 caacacagat gaatgtggct ggggagtccc catgttcatg ggccttgacc atatcataaa
     3241 gaacataacc agagagaccc tgccagctgt tctgaagaac acagcagcac ggatgatgtt
     3301 gcctccagaa tgtagtcacc tgctagtgga ggagtatatg ggggacactg aggacccaga
     3361 gaccctgcaa gcacagttca gagagatgct gggggacttc atgtttgtga tccctgctct
     3421 ccaagtggca catttccagc gttcccaggc tcctgtctac ttctatgagt tccagcatct
     3481 gtccagcttc atcaagcatg tcaggccatc acatgtgaag gcagaccatg gtgatgacgt
     3541 tgcctttgtc tttggctcct atttgtggga catgaatttg gacctcactg aggaggagga
     3601 gctgctaaag aggatgatga tgaagtactg ggccaatttc gcaagaaatg ggaaccccaa
     3661 cagtgagggt ctaccctcct ggcctgtgtt ggaccatgat gagcagtacc tgcagctgga
     3721 cacccagcct gctgtgggtc gagccctgaa ggccagaagg ctgcagttct ggaccaagac
     3781 tctgccccag aagatccagg agctcaaggg ttctcaggac aagcatcatc accaccacca
     3841 ttagtttaag ggtgggcgcg ccgacccagc tttcttgtac aaagtggttg atctagaggg
     3901 cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac
     3961 cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc
     4021 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4081 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4141 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4201 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     4261 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4321 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4381 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4441 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4501 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4561 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4621 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4681 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4741 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4801 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4861 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4921 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4981 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5041 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     5101 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     5161 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     5221 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     5281 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     5341 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     5401 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     5461 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     5521 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     5581 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     5641 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     5701 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5761 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5821 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     5881 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gcgaaatgac
     5941 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     6001 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     6061 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     6121 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     6181 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     6241 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6301 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6361 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6421 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6481 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6541 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6601 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6661 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6721 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6781 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6841 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6901 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6961 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7021 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7081 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7141 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7201 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
