LOCUS       sequence-356884-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HCVP-inducible single TM SEAP RELEASE construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  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 6981)
  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-B2AD-hcvs-KKMP
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..43)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        210..589
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        590..793
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     744..764
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    795..813
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    816..834
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     844..868
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             966..2468
                     /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             2789..2818
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2874..2891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2880..3104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3276..3295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3427..3474
                     /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             3482..4504
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3482..4504
                     /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    4634..4755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4671..4690)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4725..4744
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4804..4820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4817..4839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4828..4844
                     /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(4852..4858,4859..4876,4877..4882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4897..4918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5035..5052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5206..5794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5286..5305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5965..6756,6757..6825))
                     /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     6588..6607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6826..6930)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
       61 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
      121 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
      181 ctgcttcgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      241 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      301 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      361 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      421 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      481 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      541 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      601 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      661 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      721 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      781 tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg
      841 acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      901 ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttcg
      961 ccaccatgct gctcctgttg ctgctgctgg gcctgaggct acagctctcc ctgggcatca
     1021 tcccagttga ggaggagaac ccggacttct ggaaccgcga ggcagccgag gccctgggtg
     1081 ccgccaagaa gctgcagcct gcacagacag ccgccaagaa cctcatcatc ttcctgggcg
     1141 atgggatggg ggtgtctacg gtgacagctg ccaggattct aaaagggcag aagaaggaca
     1201 aactggggcc tgagataccc ctggccatgg accgcttccc atatgtggct ctgtccaaga
     1261 catacaatgt agacaaacat gtgccagaca gtggagccac agccacggcc tacctgtgcg
     1321 gggtcaaggg caacttccag accattggct tgagtgcagc cgcccgcttt aaccagtgca
     1381 acacgacacg cggcaacgag gtcatctccg tgatgaatcg ggccaagaaa gcagggaagt
     1441 cagtgggagt ggtaaccacc acacgagtgc agcacgcctc gccagccggc acctacgccc
     1501 acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc tgcctcggcc cgccaggagg
     1561 ggtgccagga catcgctacg cagctcatct ccaacatgga cattgacgtg atcctaggtg
     1621 gaggccgaaa atacatgttt cccatgggaa ccccagaccc tgagtaccca gatgactaca
     1681 gccaaggtgg gaccaggctg gacgggaaga atctggtgca ggaatggctg gcgaagcgcc
     1741 agggtgcccg gtatgtgtgg aaccgcactg agctcatgca ggcttccctg gacccgtctg
     1801 tgacccatct catgggtctc tttgagcctg gagacatgaa atacgagatc caccgagact
     1861 ccacactgga cccctccctg atggagatga cagaggctgc cctgcgcctg ctgagcagga
     1921 acccccgcgg cttcttcctc ttcgtggagg gtggtcgcat cgaccatggt catcatgaaa
     1981 gcagggctta ccgggcactg actgagacga tcatgttcga cgacgccatt gagagggcgg
     2041 gccagctcac cagcgaggag gacacgctga gcctcgtcac tgccgaccac tcccacgtct
     2101 tctccttcgg aggctacccc ctgcgaggga gctccatctt cgggctggcc cctggcaagg
     2161 cccgggacag gaaggcctac acggtcctcc tatacggaaa cggtccaggc tatgtgctca
     2221 aggacggcgc tcggccggat gttaccgaga gcgagagcgg gagccccgag tatcggcagc
     2281 agtcagcagt gcccctggac gaagagaccc acgcaggcga ggacgtggcg gtgttcgcgc
     2341 gcggcccgca ggcgcacctg gttcacggcg tgcaggagca gaccttcata gcgcacgtca
     2401 tggccttcgc cgcctgcctg gagccctaca ccgcctgcga cctggcaccc cccgccggca
     2461 ccaccgacgc cgcgcacccg ggtggatcct ctggcagaag caaaagaagc gtcattgacg
     2521 gctttaccct gaccagcgac gaggtgtggg tggtgggcat gggcatcgtc atgtctctca
     2581 tcgtcctggc catcgtgttt ggcaatgtgc tggtcatcac agccattgcc aagttcgagc
     2641 gtctgcagac ggtcacctct accgtgagcg aggaagctag tgaggatgtc gtctgctgct
     2701 caatgtccta cacatggaca ggagccctgg gtaccaagta caaaagcaga cgcagtttta
     2761 ttgatgaaaa gaaaatgcct tagggcccga acaaaaactc atctcagaag aggatctgaa
     2821 tagcgccgtc gaccatcatc ataatcatca ttgagtttaa acccgctgat cagcctcgac
     2881 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2941 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3001 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3061 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggagatct atcgatgttc
     3121 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3181 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3241 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3301 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3361 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3421 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
     3481 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     3541 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     3601 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     3661 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     3721 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     3781 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     3841 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     3901 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     3961 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     4021 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     4081 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     4141 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     4201 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     4261 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     4321 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     4381 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     4441 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     4501 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4561 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4621 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4681 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4741 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4801 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4861 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4981 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5041 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5101 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5161 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5221 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5281 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5341 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5401 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5461 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5521 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5581 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5641 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5701 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5761 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5821 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5881 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5941 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6001 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6061 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6121 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6181 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6241 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6301 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6361 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6421 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6481 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6541 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6601 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6661 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6721 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6781 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6841 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6901 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6961 cgacggatcg ggagatctcc c
//
