LOCUS       sequence-478443-        6645 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Expression of NLS-Kaede in Exaiptasia diaphana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6645)
  AUTHORS   n/a
  TITLE     Aiptasia Genetic Tools Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6645)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCR158
FEATURES             Location/Qualifiers
     source          1..6645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2128..2148
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2164..2841
                     /codon_start=1
                     /product="Kaede green-to-red photoconvertible fluorescent 
                     protein (Ando et al., 2002)"
                     /label=Kaede
                     /translation="MSLIKPEMKIKLLMEGNVNGHQFVIEGDGKGHPFEGKQSMDLVVK
                     EGAPLPFAYDILTTAFHYGNRVFAKYPDHIPDYFKQSFPKGFSWERSLMFEDGGVCIAT
                     NDITLKGDTFFNKVRFDGVNFPPNGPVMQKKTLKWEASTEKMYLRDGVLTGDITMALLL
                     KGDVHYRCDFRTTYKSRQEGVKLPGYHFVDHCISILRHDKDYNEVKLYEHAVAHSGLPD
                     NVK"
     polyA_signal    2856..2977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2893..2912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2947..2966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3078..3097)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3079..3097)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3104..3121)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3104..3120)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3113..3135)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3261..3689
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3348..3367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3558..3579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4033..4827
                     /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(4087..4106)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4697..4716
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4845..4913,4914..5705)
                     /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(5063..5082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5876..6464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6365..6384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6618..6635
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct ttagggataa cagggtaatg
      301 gtaccgtcga ctgcattcac tgttcttaat attgtataat aggcaaaaat cgaggtcaca
      361 gatctcgaag aaaattggta aaataaacaa aatagagaat aattaatcta atgaatcgaa
      421 aaactcgttt tcagacattc aaataagaaa caaaatgtca cataatggat ataaacaagt
      481 caatcacaat gcaaaacctc catcaccgga ggctccggtc ggattgtgtg agatgggaag
      541 ggtgcaaagg ctcatgggtg tctatagcaa gtttacacag ggatcccaaa tatatttata
      601 ctcttttaac tgtttggatt tattattctt gactttcaca tgacgtcatc agtcaaaaaa
      661 aaaatctaaa atcacagaca cacaacattg tggtactgaa atatcttgaa gagcatgata
      721 gaagttcgct ctgaatgttt aaactttttt tgctgtttat tcattgagtt attagcgttc
      781 aaaaatatgt tacgtaatac tgccacgcga cttgatacac gccaaaaata ctcgaaaagc
      841 ctatgcatct taaaacaatt atagtttcag catcaattta aatgttaaca atgatattgt
      901 tgtaaagtta aagcaaaatc atcagttttg agctcaattc gatacggtcc gccattttga
      961 agcccctgac ggggacacaa tcatggcggc cccatacatt tctttgtaaa gtaagaccgt
     1021 attcaaacgc ttacaatttt gctaggaaat cacagttttg attcaaactt tacataactg
     1081 ttgggagtta tatacttttt tgatccctga acttaaaaaa ttaatttgaa tcttaaaaca
     1141 aagctttttg tagtcatttc ttgatcagtc caaaacgcat tgagccgtgc taatcccata
     1201 cattcttacc aattttcgat cgctcgtatt tctgttaaaa tttattattt cggctcaaaa
     1261 tttcgtcatg atccttaaat taagatgttc tctcgagagc attaataaaa gtttgctaat
     1321 tctgcctcta aataggtttt ttagactatc acgtgatcag tgaaagtcaa gaattgattg
     1381 gctatgaaat tataagaaac tatgatattt attagttttt tggtaagctg aaattctaaa
     1441 gcaatcaaaa tatttttctt gtctgccagc gccatttttt tcgataagtc actttgtcga
     1501 agaaagaaga aacacctaag atagaatcta aaatcaaaag caaaaactac tagagctatg
     1561 ctaaagccaa gaagtttgca aaaaaacttg tgaatattaa aattattgaa attgaaatta
     1621 acttcggcct ttttcaggcc tttgaaaacg caaacaaaag ccagtaaatg aaccagaaaa
     1681 gacgccaaga aactaacttt gtttgctatg gtcattattc ttaattgaaa acatatgaaa
     1741 tcacaattta gcatactttt tgtctacttt tgttcggagt ctggacagcg tgctctgtca
     1801 cgctgtaagc tgtgactctt ttgttacatc tccattgttg aactgtcaag tgtttcgtag
     1861 caacccaagt cttcaatttg tgattggttt attcattctt ccgtagcgat atttaagaca
     1921 acgcattctt aatcaccgcc tcttttacat catccaaggc cacgtgtgca acttgtgagt
     1981 ttttctctta cttttactgc ccttttctcc cctttttaac tcaaattatc gttacttaag
     2041 cgaattgtga cattcgttga actgttttct ttaaaatttg tattttagat cactgttgaa
     2101 tcgctcggca acttttcaaa tatgcctcca aagaagaagc gtaaggtaga agacccagag
     2161 ctcatgagtc tgattaaacc agaaatgaag atcaagctgc ttatggaagg caatgtaaac
     2221 gggcaccagt ttgttattga gggagatgga aaaggccatc cttttgaggg aaaacagagt
     2281 atggaccttg tagtcaaaga aggcgcacct ctcccttttg cctacgatat cttgacaaca
     2341 gcattccatt atggtaacag ggtttttgct aaatacccag accatatacc agactacttc
     2401 aagcagtcgt ttcccaaagg gttttcttgg gagcgaagcc tgatgttcga ggacgggggc
     2461 gtttgcatcg ctacaaatga cataacactg aaaggagaca ctttttttaa caaagttcga
     2521 tttgatggcg taaactttcc cccaaatggt cctgttatgc agaagaagac tctgaaatgg
     2581 gaggcatcca ctgagaaaat gtatttgcgt gatggagtgt tgacgggcga tattaccatg
     2641 gctctgctgc ttaaaggaga tgtccattac cgatgtgact tcagaactac ttacaaatct
     2701 aggcaggagg gtgtcaagtt gccaggatat cactttgtcg atcactgcat cagcatattg
     2761 aggcatgaca aagactacaa cgaggttaag ctgtatgagc atgctgttgc ccattctgga
     2821 ttgccggaca acgtcaagta gcaattgttg ttgttaactt gtttattgca gcttataatg
     2881 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2941 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaaag ggccggccat taccctgtta
     3001 tccctatcaa gggcgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat
     3061 ctagagggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac
     3121 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3181 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3241 gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3301 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3361 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3421 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3481 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3541 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3601 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3661 gctgatttaa caaaaattta acgcgaattt taacaaaatt cagggcgcaa gggctgctaa
     3721 aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     3781 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     3841 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     3901 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     3961 ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga
     4021 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4081 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4141 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4201 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4261 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4321 gtgccggggc aggatctcct gtcatcccac cttgctcctg ccgagaaagt atccatcatg
     4381 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4441 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4501 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4561 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4621 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4681 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4741 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4801 tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt attcaacatt
     4861 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4921 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4981 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5041 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5101 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5161 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5221 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5281 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5341 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5401 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5461 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5521 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5581 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5641 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5701 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5761 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5821 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5881 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5941 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6001 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6061 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6121 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6181 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6241 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6301 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6361 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6421 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6481 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6541 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6601 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
