LOCUS       sequence-459263-        6471 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Membrane-bound human-to-mouse (H2M) renin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  AUTHORS   Aldrete CA, Call CC, Sant'Anna LE, Vlahos AE, Pei J, Cong Q, Gao XJ
  TITLE     Orthogonalized human protease control of secreted signals.
  JOURNAL   Nat Chem Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x.
  PUBMED    39814991
REFERENCE   2  (bases 1 to 6471)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x."
COMMENT     Sequence Label: CA-0151 CMVTO-Renin(H2M)-ECL
FEATURES             Location/Qualifiers
     source          1..6471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     265..284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(503..607)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(682..701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        868..1247
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1248..1451
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1402..1422
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1453..1471
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1474..1492
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1502..1526
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1618..1627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2937..2966
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3022..3039)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3028..3252
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3424..3443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3575..3622
                     /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             3630..4652
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3630..4652
                     /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    4782..4903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4819..4838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4873..4892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4952..4968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4952..4968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4965..4987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4976..4992
                     /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(5000..5006,5007..5024,5025..5030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5045..5066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5183..5200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5354..5942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5434..5453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6113..6471,1..433,434..502))
                     /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"
ORIGIN
        1 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
       61 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
      121 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
      181 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
      241 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
      301 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
      361 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
      421 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
      481 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
      541 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
      601 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
      661 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
      721 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      781 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      841 gcttcgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      901 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      961 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1021 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1081 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1141 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1201 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1261 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1321 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1381 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1441 taagcagagc tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac
     1501 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     1561 atagaagaca ccgggaccga tccagcctcc ggactctagc gtttaaactt aagcttcgcc
     1621 accatggacg gttggagaag gatgccccgg tggggccttt tgctgctcct gtggggaagc
     1681 tgcaccttcg gggctagcct cactctcggt aatactactt cctcagtcat cttaaccaac
     1741 tacatggata cgcagtatta cggggaaatc ggcattggca caccccctca gacttttaag
     1801 gtagtttttg atactgggtc tagcaatgtt tgggtcccta gcagtaagtg ttcccgcctg
     1861 tatacagcgt gtgtttacca taagttattt gacgcctcag actcctctag ctataaacac
     1921 aacggcaccg agctcaccat ccactacagc acagggactg tttcggggtt tctgtcccag
     1981 gacattatta cagtgggagg tattacagtg acccaaatgt ttggggaggt gaccgagatg
     2041 ccggctctgc cgttcatgct ggcagagttc gacggggtcg ttggcatggg cttcatcgag
     2101 caggctgtgg ggcgggtaac ccccatcttc gacaacatca ttagccaggg cgtgctaaag
     2161 gaggacgtgt tcagctttta ttacaataga gattctgaaa actctcagag tttgggcggc
     2221 cagatagtgc ttggaggcag tgatccccag cactacgaag gcaattttca ctatataaat
     2281 ctgatcaaaa ctggggtgtg gcagatccag atgaaggggg tgtcagtggg ttcctctacg
     2341 ttattgtgcg aggacggatg tctggccgtg gtagacactg gagctagtta catcagcggc
     2401 tcaaccagct caattgaaaa gctgatggag gcactgggag caaaaaagcg gttgttcgat
     2461 tacgtcgtga aatgtaatga gggacctacc ctgcccgaca tctctttcca tctgggtgga
     2521 aaagagtata ctctgacaag cgccgattac gtatttcagg agtcctactc gtccaaaaaa
     2581 ctctgcacgt tagcaatcca cgccatggac atccctccac caaccggacc tacctgggct
     2641 ctgggggcca catttatacg aaaattctat accgagttcg atcgcaggaa caaccgtatc
     2701 ggctttgcac ttgccagggg cgccgggaca ggagggtctg ggggtggcac tggcggatct
     2761 ggaggagact acaaggacga tgacgacaac ggcggatcgg gggggtctgg tggtagtggc
     2821 ggttcggggg gaggcacagg tggatccctg gtcgtggttg ctggagtcct gttttgttat
     2881 ggcttgctag tgacagtggc tctttgtgtt gaaaacgggg gcggctctta gggcccgaac
     2941 aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat aatcatcatt
     3001 gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     3061 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     3121 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3181 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3241 tgggctctat ggagatctat cgatgttctg gggatgcggt gggctctatg gcttctgagg
     3301 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3361 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3421 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3481 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3541 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3601 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3661 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3721 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3781 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3841 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     3901 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3961 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     4021 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4081 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4141 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4201 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4261 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4321 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4381 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4441 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4501 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4561 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4621 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4681 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4741 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4801 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4861 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4921 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4981 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5041 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5101 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5161 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5221 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5281 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5341 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5401 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5461 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5521 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5581 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5641 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5701 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5761 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5821 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5881 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5941 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6001 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6061 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6121 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6181 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6241 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6301 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6361 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6421 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca t
//
