LOCUS       sequence-479207-        7422 bp    DNA     circular SYN 31-JUL-2025
DEFINITION  Expresses RELEASE-LATeNT cargo in mammalian cells; used with 
            cytsolic LATeNT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7422)
  AUTHORS   Roh H, Kim D, Kim B, Jeon Y, Kim Y, Jacko M, Xu F, Lin C, Won Um J, 
            Ting AY
  TITLE     Light-activated tetanus neurotoxin for conditional proteolysis and 
            inducible synaptic inhibition in vivo
  JOURNAL   bioRxiv 2025.01.27.635161; doi: 10.1101/2025.01.27.635161
REFERENCE   2  (bases 1 to 7422)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 31, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.01.27.635161; doi: 10.1101/2025.01.27.635161"
COMMENT     Sequence Label: pcDNA3-RELEASE-LATeNT
FEATURES             Location/Qualifiers
     source          1..7422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        167..546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        547..750
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     701..721
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    752..770
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    773..791
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     801..825
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             923..2425
                     /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             3187..3216
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3272..3289)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3278..3502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3674..3693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3825..3872
                     /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             3880..4902
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3880..4902
                     /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    5032..5153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5069..5088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5123..5142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5202..5218)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5202..5218)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5215..5237)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5226..5242
                     /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(5250..5256,5257..5274,5275..5280))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5295..5316
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5433..5450)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5604..6192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5684..5703)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6363..7154,7155..7223))
                     /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     6986..7005
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7224..7328)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7403..7422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
       61 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      121 gggttaggcg ttttgcgctg cttcgtacgg gccagatata cgcgttgaca ttgattattg
      181 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      241 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      301 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      361 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      421 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      481 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      541 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      601 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      661 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      721 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
      781 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
      841 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
      901 tttaaactta agcttcgcca ccatgctgct cctgttgctg ctgctgggcc tgaggctaca
      961 gctctccctg ggcatcatcc cagttgagga ggagaacccg gacttctgga accgcgaggc
     1021 agccgaggcc ctgggtgccg ccaagaagct gcagcctgca cagacagccg ccaagaacct
     1081 catcatcttc ctgggcgatg ggatgggggt gtctacggtg acagctgcca ggattctaaa
     1141 agggcagaag aaggacaaac tggggcctga gatacccctg gccatggacc gcttcccata
     1201 tgtggctctg tccaagacat acaatgtaga caaacatgtg ccagacagtg gagccacagc
     1261 cacggcctac ctgtgcgggg tcaagggcaa cttccagacc attggcttga gtgcagccgc
     1321 ccgctttaac cagtgcaaca cgacacgcgg caacgaggtc atctccgtga tgaatcgggc
     1381 caagaaagca gggaagtcag tgggagtggt aaccaccaca cgagtgcagc acgcctcgcc
     1441 agccggcacc tacgcccaca cggtgaaccg caactggtac tcggacgccg acgtgcctgc
     1501 ctcggcccgc caggaggggt gccaggacat cgctacgcag ctcatctcca acatggacat
     1561 tgacgtgatc ctaggtggag gccgaaaata catgtttccc atgggaaccc cagaccctga
     1621 gtacccagat gactacagcc aaggtgggac caggctggac gggaagaatc tggtgcagga
     1681 atggctggcg aagcgccagg gtgcccggta tgtgtggaac cgcactgagc tcatgcaggc
     1741 ttccctggac ccgtctgtga cccatctcat gggtctcttt gagcctggag acatgaaata
     1801 cgagatccac cgagactcca cactggaccc ctccctgatg gagatgacag aggctgccct
     1861 gcgcctgctg agcaggaacc cccgcggctt cttcctcttc gtggagggtg gtcgcatcga
     1921 ccatggtcat catgaaagca gggcttaccg ggcactgact gagacgatca tgttcgacga
     1981 cgccattgag agggcgggcc agctcaccag cgaggaggac acgctgagcc tcgtcactgc
     2041 cgaccactcc cacgtcttct ccttcggagg ctaccccctg cgagggagct ccatcttcgg
     2101 gctggcccct ggcaaggccc gggacaggaa ggcctacacg gtcctcctat acggaaacgg
     2161 tccaggctat gtgctcaagg acggcgctcg gccggatgtt accgagagcg agagcgggag
     2221 ccccgagtat cggcagcagt cagcagtgcc cctggacgaa gagacccacg caggcgagga
     2281 cgtggcggtg ttcgcgcgcg gcccgcaggc gcacctggtt cacggcgtgc aggagcagac
     2341 cttcatagcg cacgtcatgg ccttcgccgc ctgcctggag ccctacaccg cctgcgacct
     2401 ggcacccccc gccggcacca ccgacgccgc gcacccgggt ggatcctctg gcagaagcaa
     2461 aagaagcgtc attgacggct ttaccctgac cagcgacgag gtgtgggtgg tgggcatggg
     2521 catcgtcatg tctctcatcg tcctggccat cgtgtttggc aatgtgctgg tcatcacagc
     2581 cattgccaag ttcgagcgtc tgcagacggt caccgggggc ggctctctcg aggggggggg
     2641 ctcggggctc tcggctaccg ctgccaccgt cccgcctgcc gccccggccg gcgagggtgg
     2701 cccccctgca cctcctccaa accttactag taacaggaga ctgcagcaga cccaggccca
     2761 ggtggatgag gtggtggaca tcatgagggt gaatgtggac aaggtcctgg agcgggacca
     2821 gaagttgtcg gagctggatg accgtgcaga tgccctccag gcaggggcct cccagtttga
     2881 aacaagtgca gccaagctca agcgcaaata ctggtggaaa aacctcaagg gcagtggagg
     2941 taccaattac ttcatcactt cactggcctg tgctgatctg gtcatgggcc tggcagtggt
     3001 gccctttggg gccgcccata ttcttatgaa aatgtggact tttggcaact tctgggatat
     3061 ctacatctgg gctcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac
     3121 cctttactgc aacaagtaca aaagcagacg cagttttatt gatgaaaaga aaatgcctta
     3181 gggcccgaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat
     3241 aatcatcatt gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     3301 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3361 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3421 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3481 ggggatgcgg tgggctctat ggagatctat cgatgttctg gggatgcggt gggctctatg
     3541 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3601 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3661 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3721 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3781 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     3841 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     3901 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3961 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     4021 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     4081 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     4141 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     4201 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     4261 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     4321 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     4381 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     4441 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     4501 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     4561 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     4621 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     4681 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4741 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     4801 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     4861 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     4921 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4981 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5041 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5101 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5161 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5221 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5281 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5341 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5401 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5461 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5521 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5581 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5641 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5701 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5761 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5821 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5881 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5941 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6001 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6061 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6121 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6181 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6241 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6301 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6361 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6421 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6481 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6541 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6601 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6661 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6721 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6781 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6841 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6901 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6961 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7021 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7081 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7141 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7201 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7261 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7321 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     7381 atcccctatg gtgcactctc agtacaatct gctctgatgc cg
//
