LOCUS       sequence-356887-        7164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HCVP-inducible tri-transmembrane SEAP RELEASE construct.
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-hcvs-KKMP
FEATURES             Location/Qualifiers
     source          1..7164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    695..713
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    716..734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     744..768
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             866..2368
                     /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             2872..2901
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2957..2974)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2963..3187
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3359..3378)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3510..3557
                     /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             3565..4587
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3565..4587
                     /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    4717..4838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4754..4773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4808..4827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4887..4903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4900..4922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4911..4927
                     /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(4935..4941,4942..4959,4960..4965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4980..5001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5118..5135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5289..5877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5369..5388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6048..6839,6840..6908))
                     /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     6671..6690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6909..7013)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7088..7107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct
      721 atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc
      781 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccggactcta
      841 gcgtttaaac ttaagcttcg ccaccatgct gctcctgttg ctgctgctgg gcctgaggct
      901 acagctctcc ctgggcatca tcccagttga ggaggagaac ccggacttct ggaaccgcga
      961 ggcagccgag gccctgggtg ccgccaagaa gctgcagcct gcacagacag ccgccaagaa
     1021 cctcatcatc ttcctgggcg atgggatggg ggtgtctacg gtgacagctg ccaggattct
     1081 aaaagggcag aagaaggaca aactggggcc tgagataccc ctggccatgg accgcttccc
     1141 atatgtggct ctgtccaaga catacaatgt agacaaacat gtgccagaca gtggagccac
     1201 agccacggcc tacctgtgcg gggtcaaggg caacttccag accattggct tgagtgcagc
     1261 cgcccgcttt aaccagtgca acacgacacg cggcaacgag gtcatctccg tgatgaatcg
     1321 ggccaagaaa gcagggaagt cagtgggagt ggtaaccacc acacgagtgc agcacgcctc
     1381 gccagccggc acctacgccc acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc
     1441 tgcctcggcc cgccaggagg ggtgccagga catcgctacg cagctcatct ccaacatgga
     1501 cattgacgtg atcctaggtg gaggccgaaa atacatgttt cccatgggaa ccccagaccc
     1561 tgagtaccca gatgactaca gccaaggtgg gaccaggctg gacgggaaga atctggtgca
     1621 ggaatggctg gcgaagcgcc agggtgcccg gtatgtgtgg aaccgcactg agctcatgca
     1681 ggcttccctg gacccgtctg tgacccatct catgggtctc tttgagcctg gagacatgaa
     1741 atacgagatc caccgagact ccacactgga cccctccctg atggagatga cagaggctgc
     1801 cctgcgcctg ctgagcagga acccccgcgg cttcttcctc ttcgtggagg gtggtcgcat
     1861 cgaccatggt catcatgaaa gcagggctta ccgggcactg actgagacga tcatgttcga
     1921 cgacgccatt gagagggcgg gccagctcac cagcgaggag gacacgctga gcctcgtcac
     1981 tgccgaccac tcccacgtct tctccttcgg aggctacccc ctgcgaggga gctccatctt
     2041 cgggctggcc cctggcaagg cccgggacag gaaggcctac acggtcctcc tatacggaaa
     2101 cggtccaggc tatgtgctca aggacggcgc tcggccggat gttaccgaga gcgagagcgg
     2161 gagccccgag tatcggcagc agtcagcagt gcccctggac gaagagaccc acgcaggcga
     2221 ggacgtggcg gtgttcgcgc gcggcccgca ggcgcacctg gttcacggcg tgcaggagca
     2281 gaccttcata gcgcacgtca tggccttcgc cgcctgcctg gagccctaca ccgcctgcga
     2341 cctggcaccc cccgccggca ccaccgacgc cgcgcacccg ggtggatcct ctggcagaag
     2401 caaaagaagc gtcattgacg gctttaccct gaccagcgac gaggtgtggg tggtgggcat
     2461 gggcatcgtc atgtctctca tcgtcctggc catcgtgttt ggcaatgtgc tggtcatcac
     2521 agccattgcc aagttcgagc gtctgcagac ggtcacctct accgtgagcg aggaagctag
     2581 tgaggatgtc gtctgctgct caatgtccta cacatggaca ggagccctga attacttcat
     2641 cacttcactg gcctgtgctg atctggtcat gggcctggca gtggtgccct ttggggccgc
     2701 ccatattctt atgaaaatgt ggacttttgg caacttctgg gatatctaca tctgggctcc
     2761 cttggccggg acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaacaa
     2821 gtacaaaagc agacgcagtt ttattgatga aaagaaaatg ccttagggcc cgaacaaaaa
     2881 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcataatca tcattgagtt
     2941 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3001 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3061 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3121 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3181 tctatggaga tctatcgatg ttctggggat gcggtgggct ctatggcttc tgaggcggaa
     3241 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3301 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3361 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3421 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3481 cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta
     3541 gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     3601 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     3661 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     3721 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     3781 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     3841 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     3901 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     3961 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     4021 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     4081 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     4141 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     4201 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     4261 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     4321 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     4381 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     4441 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     4501 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     4561 agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc
     4621 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4681 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4741 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4801 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4861 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4921 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4981 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5041 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5101 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5161 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5221 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5281 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5341 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5401 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5461 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5521 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5581 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5641 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5701 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5761 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5821 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5881 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5941 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6001 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6061 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6121 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6181 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6241 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6301 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6361 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6421 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6481 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6541 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6601 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6661 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6721 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6781 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6841 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6901 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6961 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7021 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     7081 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     7141 tgttggaggt cgctgagtag tgcg
//
