LOCUS       sequence-487595-        6086 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Expresses KORD-NanoLuc in mammalian cells; used with 
            LATeNT*-Arrestin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6086)
  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 6086)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 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: pAAV-KORD-NanoLuc
FEATURES             Location/Qualifiers
     source          1..6086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..135
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     758..777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        758..776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     796..855
                     /codon_start=1
                     /product="human interleukin-2 signal sequence"
                     /label=IL-2 signal sequence
                     /translation="MYRMQLLSCIALSLALVTNS"
     CDS             1264..1302
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             2524..3033
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             3034..3075
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3112..3129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3118..3325
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3354..3494
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3354..3483
                     /label=AAV2 ITR
     rep_origin      3569..4024
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3656..3675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3866..3887
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4041..4060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4160..4182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4306..4410
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4411..4479,4480..5271)
                     /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     complement(4629..4648)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5442..6030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5931..5950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
       61 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      121 tcactagggg ttcctgcggc cgcacgcgtt gtacatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      721 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      781 cccaagcttg ccaccatgta caggatgcaa ctcctgtctt gcattgcact aagtcttgca
      841 cttgtcacga attcggtgca actcgtagag tccggtggcg gactcgtcca ggcgggaggc
      901 tccctgcgcc tgagttgcgc tgcgtccgga agaactatta gcatggccgc catgagttgg
      961 ttcagacagg cgcctggaaa ggagagggag ttcgtggcag gcataagtcg ctccgcaggg
     1021 tcagccgtcc atgccgactc cgtaaaggga cgctttacca tctccagaga taacacaaaa
     1081 aacacgctct atctccaaat gaattctttg aaggccgagg acacagccgt gtactactgc
     1141 gccgttagga ccagcggatt ttttggaagc attcctagga cagggacagc ctttgattac
     1201 tggggccagg gaacgcaggt taccgtttcc accggtggtg gcggctccgg tggagggtct
     1261 ccgtccagac ttgaggaaga gttgagacgg aggttgaccg agccaggcgg tggcagtgga
     1321 ggtgggtccg gtaccatgga aagccccatt caaatcttcc ggggcgagcc cggaccaacc
     1381 tgcgctccaa gcgcttgcct gccaccgaat tctagtgcat ggttcccagg gtgggcagag
     1441 cccgatagca atggctcagc cgggtccgag gatgcgcagc ttgaacccgc tcacatctca
     1501 cccgctatcc ctgtcattat tacggcagtc tactcagttg ttttcgttgt gggcctggtt
     1561 ggaaatagcc tcgtgatgtt tgtgattatc aggtatacta aaatgaagac cgccacgaat
     1621 atctacatct ttaacctggc tctggctgac gccttggtga ccaccactat gccatttcag
     1681 tccaccgtct atcttatgaa ctcttggcca ttcggcgacg ttctgtgcaa gattgtcatt
     1741 tccattaatt attataatat gtttacatca atctttacgc ttacaatgat gagcgtggac
     1801 cgatacattg ctgtttgcca tccggtgaaa gcactggatt ttcgcacccc cttgaaggcc
     1861 aagattatca atatctgtat ttggctcctg tcctcatccg tcggaatttc cgccattgtc
     1921 ttgggtggta caaaggtgag ggaggacgtg gacgttatag agtgcagcct gcagttccct
     1981 gacgatgact acagctggtg ggaccttttt atgaagatct gtgtcttcat ttttgctttc
     2041 gtcatccccg tgctgattat catagtgtgc tatacactga tgatattgcg gctgaaatct
     2101 gtgcgcctgc tgagcggcag ccgcgaaaag gatcggaatc tgcgccgaat cacacgcctc
     2161 gtgctggttg tagtcgctgt cttcgtggta tgctggaccc caatccacat tttcatcctg
     2221 gtggaagctc tgggatccac ctctcactcc actgccgcac ttagctcata ttacttctgc
     2281 attgccttgg gctacactaa ttcctcactg aatcccattc tgtacgcatt tcttgacgag
     2341 aacttcaaac ggtgctttag agacttttgc ttccccctca agatgcgcat ggagcggcag
     2401 tcaaccagcc gagtccggaa tacggtccag gatcccgcct atctgcggga catcgacggc
     2461 atgaacaagc ctgtggagct ggccgagaag ctggccggcc tggacatcaa cggtggagct
     2521 agcgtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg
     2581 gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta
     2641 actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc
     2701 atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag
     2761 gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta
     2821 atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc
     2881 gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc
     2941 gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg
     3001 accggctggc ggctgtgcga acgcattctg gcgggcaagc cgatccctaa ccctctgctg
     3061 ggcctggaca gcacttagta agaattcctc gagctagagc tcgctgatca gcctcgactg
     3121 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3181 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3241 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3301 aagacaatag caggcatgct ggggaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     3361 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3421 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3481 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3541 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     3601 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     3661 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3721 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3781 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3841 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3901 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3961 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4021 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4081 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4141 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4201 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4261 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4321 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4381 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4441 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4501 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4561 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4621 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4681 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4741 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4801 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4861 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4921 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4981 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5041 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5101 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     5161 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5221 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5281 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5341 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5401 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5461 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5521 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5581 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5641 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5701 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5761 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5821 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5881 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5941 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6001 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6061 ctggcctttt gctggccttt tgctca
//
