LOCUS       sequence-478911-        7820 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expresses NanoLuc-LATeNT in the mammalian cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7820)
  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 7820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 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-NanoLuc-LATeNT
FEATURES             Location/Qualifiers
     source          1..7820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..951
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             988..1497
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     promoter        complement(3373..3391)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3374..3391)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3411..3428)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3417..3641
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3687..4115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3774..3793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3984..4005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4124..4144)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4129..4458
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4309..4444
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4371..4390
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4525..5319
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4579..4598)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5189..5208
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5493..5614
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5530..5549)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5584..5603
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5663..5679)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5663..5679)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5676..5698)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5687..5703
                     /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(5711..5717,5718..5735,5736..5741))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5756..5777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5894..5911)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6065..6653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6145..6164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6824..7615,7616..7684))
                     /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     7447..7466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7685..7789)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg gcaagcccat ccccaacccc ctgctgggcc tggacagcac caagggctcg
      961 ggctcgacct cgggctcggg caccggtgtc ttcacactcg aagatttcgt tggggactgg
     1021 cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg
     1081 tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat
     1141 gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg
     1201 ggccagatcg aaaaaatttt taaggtggtg taccctgtgg atgatcatca ctttaaggtg
     1261 atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc
     1321 ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc
     1381 ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg
     1441 ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcgggg
     1501 agcggcagcg gggggtccgg tagcgggccg atcaccatca acaacttccg ttactccgac
     1561 ccggttaaca acgacaccat catcatgatg gaaccgccgt actgcaaagg tctagacatc
     1621 tactacagag ccttcaaaat caccgaccgt atctggatcg ttccggaacg ttacgaattt
     1681 ggtaccaaac cggaagactt caacccgccg tcctccctga tcgaaggtgc ttccgaatac
     1741 tacgacccga actacctgcg taccgactcc gacaaagacc gtttcctgca gaccatggtt
     1801 aaactgttca accgtatcaa aaacaacgtt gctggtgaag ctctgctgga caaaatcatc
     1861 aacgctatcc cgtacctggg taactcctac tccctgctgg acaaattcga caccaactcc
     1921 aactccgttt ccttcaacct gctggaacag gatccgtccg gtgctaccac caaatccgct
     1981 atgctgacca acctgatcat cttcggtccg ggtccggttc tgaacaaaaa cgaagttcgt
     2041 ggtatcgttc tgcgtgttga caacaaaaac tacttcccgt gccgtgacgg tttcggttcc
     2101 atcatgcaga tggctttctg cccggaatac gtaccgacct tcgacaacgt tatcgaaaac
     2161 atcacctccc tgaccatcgg taaatccaaa tacttccagg acccggctct gctgctgatg
     2221 cacgaactga tccacgttct gcacggtctg tacggcatgc aggtttcctc ccacgaaatc
     2281 atcccgtcca aacaggaaat ctacatgcag cacacctacc cgatctccgc tgaagagctc
     2341 ttcaccttcg gtggtcagga cgctaacctg atctccatcg atatcaaaaa cgacctgtac
     2401 gaaaaaaccc tgaacgacta caaagctatc gctaacaaac tgtcccaggt tacctcctgc
     2461 aacgacccga acatcgacat cgactcctac aaacagatct atcagcagaa gtatcagttt
     2521 gataaggatt ccaacggcca gtatatagtg aacgaggata agtttcagat actgtataac
     2581 tccataatgt atggcgggag cggcagcggg gggtccggta gcggggagtt tcgggcaacc
     2641 acactggaac ggatcgagaa aagcttcgtg attactgatc cgagactgcc tgacaaccca
     2701 atcatttttg tgagcaattc cttcctgcag ctgacagaat attctcggga agagatcctg
     2761 gggcgcaatt gccgttttct gcagggaccc gagacagacc gtgccactgt tcggaaaatc
     2821 agagatgcta ttgacaacca gactgaagtg accgttcagc tgatcaatta taccaagagc
     2881 ggcaagaagt tctggaacgt gttccacctg cagccgatgc gcgattataa gggcgacgtc
     2941 cagtacttca ttggcgtgca gctggatggc accgaacgcc ttcatggcgc cgctgagcgt
     3001 gaggcggtct gcctggtcaa aaagacagcc tttcagattt ttactgagat agagctgggc
     3061 aagaagttta acataaagac tcgcttaagc tatttttcca tgaaccacga tcctgtgaag
     3121 atacctaacc tgctggatga tactatatat aacgatactg agggctttaa catagagtcc
     3181 aaggatctga agtccgagta taagggccag aacatgaggg tgaacactaa cgcctttagg
     3241 aacgtcgacg gttccggtct ggtttccaaa ctgatcggtc tgtgcaaaaa aatcatcccg
     3301 ccgaccaaca tccgtgaaaa cctgtacaac cgtaccgctt aataactcga gcatgcatct
     3361 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     3421 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3481 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3541 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3601 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3661 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3721 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3781 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3841 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3901 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3961 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4021 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4081 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4141 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4201 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4261 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4321 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4381 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4441 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4501 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4561 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4621 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4681 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4741 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4801 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4861 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4921 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4981 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     5041 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5101 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5161 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5221 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5281 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5341 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5401 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5461 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5521 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5581 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5641 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5701 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5761 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5821 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5881 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5941 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6001 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6061 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6121 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6181 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6241 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6301 gctccaagct gggctgtgtg cacaaacccc ccgttcagcc cgaccgctgc gccttatccg
     6361 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6421 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6481 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6541 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6601 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6661 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6721 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6781 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6841 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6901 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6961 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7021 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7081 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7141 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7201 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7261 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7321 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7381 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7441 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7501 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7561 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7621 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7681 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7741 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7801 gaaaagtgcc acctgacgtc
//
