LOCUS       sequence-356789-        7164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SEAP RELEASE construct with AAMP motif control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7164)
  AUTHORS   Vlahos AE, Kang J, Aldrete CA, Zhu R, Chong LS, Elowitz MB, Gao XJ
  TITLE     Protease-controlled secretion and display of intercellular signals.
  JOURNAL   Nat Commun. 2022 Feb 17;13(1):912. doi: 10.1038/s41467-022-28623-y.
  PUBMED    35177637
REFERENCE   2  (bases 1 to 7164)
  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: "Nat 
            Commun."; date: "2022-02-17"; volume: "13(1):912. doi"; pages: " 
            10.1038/s41467-022-28623-y"
COMMENT     Sequence Label: CMVTO-SEAP-26Sfur-3TM-tevs-AAMP
FEATURES             Location/Qualifiers
     source          1..7164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    675..693
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    696..714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     724..748
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             846..2348
                     /codon_start=1
                     /product="secreted alkaline phosphatase from human 
                     placenta"
                     /label=SEAP
                     /note="The natural enzyme has a C-terminal transmembrane 
                     sequence that is replaced by a GPI anchor."
                     /translation="MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQP
                     AQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDK
                     HVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVV
                     TTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRK
                     YMFPMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTH
                     LMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESR
                     AYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKA
                     RDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFA
                     RGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD"
     CDS             2574..2594
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2852..2881
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2937..2954)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2943..3167
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3339..3358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3490..3537
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3545..4567
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3545..4567
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4697..4818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4734..4753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4788..4807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4867..4883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4880..4902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4891..4907
                     /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(4915..4921,4922..4939,4940..4945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4960..4981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5098..5115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5269..5857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5349..5368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6028..6819,6820..6888))
                     /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     6651..6670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6889..6993)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7068..7087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg
      721 acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      781 ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttcg
      841 ccaccatgct gctcctgttg ctgctgctgg gcctgaggct acagctctcc ctgggcatca
      901 tcccagttga ggaggagaac ccggacttct ggaaccgcga ggcagccgag gccctgggtg
      961 ccgccaagaa gctgcagcct gcacagacag ccgccaagaa cctcatcatc ttcctgggcg
     1021 atgggatggg ggtgtctacg gtgacagctg ccaggattct aaaagggcag aagaaggaca
     1081 aactggggcc tgagataccc ctggccatgg accgcttccc atatgtggct ctgtccaaga
     1141 catacaatgt agacaaacat gtgccagaca gtggagccac agccacggcc tacctgtgcg
     1201 gggtcaaggg caacttccag accattggct tgagtgcagc cgcccgcttt aaccagtgca
     1261 acacgacacg cggcaacgag gtcatctccg tgatgaatcg ggccaagaaa gcagggaagt
     1321 cagtgggagt ggtaaccacc acacgagtgc agcacgcctc gccagccggc acctacgccc
     1381 acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc tgcctcggcc cgccaggagg
     1441 ggtgccagga catcgctacg cagctcatct ccaacatgga cattgacgtg atcctaggtg
     1501 gaggccgaaa atacatgttt cccatgggaa ccccagaccc tgagtaccca gatgactaca
     1561 gccaaggtgg gaccaggctg gacgggaaga atctggtgca ggaatggctg gcgaagcgcc
     1621 agggtgcccg gtatgtgtgg aaccgcactg agctcatgca ggcttccctg gacccgtctg
     1681 tgacccatct catgggtctc tttgagcctg gagacatgaa atacgagatc caccgagact
     1741 ccacactgga cccctccctg atggagatga cagaggctgc cctgcgcctg ctgagcagga
     1801 acccccgcgg cttcttcctc ttcgtggagg gtggtcgcat cgaccatggt catcatgaaa
     1861 gcagggctta ccgggcactg actgagacga tcatgttcga cgacgccatt gagagggcgg
     1921 gccagctcac cagcgaggag gacacgctga gcctcgtcac tgccgaccac tcccacgtct
     1981 tctccttcgg aggctacccc ctgcgaggga gctccatctt cgggctggcc cctggcaagg
     2041 cccgggacag gaaggcctac acggtcctcc tatacggaaa cggtccaggc tatgtgctca
     2101 aggacggcgc tcggccggat gttaccgaga gcgagagcgg gagccccgag tatcggcagc
     2161 agtcagcagt gcccctggac gaagagaccc acgcaggcga ggacgtggcg gtgttcgcgc
     2221 gcggcccgca ggcgcacctg gttcacggcg tgcaggagca gaccttcata gcgcacgtca
     2281 tggccttcgc cgcctgcctg gagccctaca ccgcctgcga cctggcaccc cccgccggca
     2341 ccaccgacgc cgcgcacccg ggtggatcct ctggcagaag caaaagaagc gtcattgacg
     2401 gctttaccct gaccagcgac gaggtgtggg tggtgggcat gggcatcgtc atgtctctca
     2461 tcgtcctggc catcgtgttt ggcaatgtgc tggtcatcac agccattgcc aagttcgagc
     2521 gtctgcagac ggtcaccggg ggcggctctc tcgagggggg gggctcgggg ctcgaaaact
     2581 tgtacttcca gagtggcagt ggaggtacca attacttcat cacttcactg gcctgtgctg
     2641 atctggtcat gggcctggca gtggtgccct ttggggccgc ccatattctt atgaaaatgt
     2701 ggacttttgg caacttctgg gatatctaca tctgggctcc cttggccggg acttgtgggg
     2761 tccttctcct gtcactggtt atcacccttt actgcaacaa gtacaaaagc agacgcagtt
     2821 ttattgatga agccgccatg ccttagggcc cgaacaaaaa ctcatctcag aagaggatct
     2881 gaatagcgcc gtcgaccatc atcataatca tcattgagtt taaacccgct gatcagcctc
     2941 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3001 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3061 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3121 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaga tctatcgatg
     3181 ttctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3241 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3301 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3361 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3421 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3481 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     3541 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     3601 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     3661 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     3721 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     3781 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     3841 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     3901 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     3961 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     4021 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     4081 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     4141 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     4201 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     4261 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     4321 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     4381 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     4441 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     4501 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     4561 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4621 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4681 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4861 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4921 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4981 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5041 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5101 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5161 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5221 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5281 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5341 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5401 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5461 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5521 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5581 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5641 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5701 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5761 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5821 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5881 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5941 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6001 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6061 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6121 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6181 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6241 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6301 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6361 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6421 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6481 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6541 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6601 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6661 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6721 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6781 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6841 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6901 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6961 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7021 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7081 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     7141 tgcgcgagca aaatttaagc taca
//
