LOCUS       sequence-430295-        5854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli leucine tRNA variant ""LeuIGI2"" and a wild-type 
            mCherry reporter; can be packaged into AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5854)
  AUTHORS   Huang RL, Jewel D, Kelemen RE, Pham Q, Yared TJ, Wang S, Singha Roy 
            SJ, Huang Z, Levinson SD, Sundaresh B, Miranda SE, van Opijnen T, 
            Chatterjee A
  TITLE     Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for
            Enhanced Noncanonical Amino Acid Mutagenesis.
  JOURNAL   ACS Synth Biol. 2024 Jul 19;13(7):2141-2149. doi: 
            10.1021/acssynbio.4c00196. Epub 2024 Jun 21.
  PUBMED    38904157
REFERENCE   2  (bases 1 to 5854)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.4c00196"; journalName: "ACS Synth Biol"; date: 
            "2024-07-19- 19"; volume: "13"; issue: "7"; pages: "2141-2149"
COMMENT     Sequence Label: pAAV-ITR-CMV-mCherry-1xU6-LeuIGI2
FEATURES             Location/Qualifiers
     source          1..5854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        212..460
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     212..232
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     383..402
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        724..1027
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1028..1231
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1182..1202
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1228..1252
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1380..1758
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1836..2546
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1981..1999)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2249..2268
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2601..3077
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2800..2819)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3117..3257
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3117..3246
                     /label=AAV2 ITR
     rep_origin      3332..3787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3419..3438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3629..3650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3804..3823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3923..3945
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3983..4001)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4069..4173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4174..4242,4243..5034)
                     /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(4392..4411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5205..5793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5694..5713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctcgcg gtccagtagt gatcgacact gctcgatccg
      181 ctcgcacccc taggggtacc tcgggcagga agagggccta tttcccatga ttccttcata
      241 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      301 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      361 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      421 tttcttggct ttatatatct tgtggaaagg acgaaacacc gggcgtgtgg tggaatcggt
      481 agacacaagg gattctaaat ccctcggcgt tcgcgctgtg cgggttcaag tcccgccgcg
      541 cccatttttt gctagcggat cgacgagagc agcgcgactg gatctgtcgc ccgtctcaaa
      601 cgcaaccctc cggcggtcgc atatcattca ggacgagcct cagactccag cgtaacacgc
      661 gtctagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      721 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      781 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      841 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      901 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      961 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1021 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1081 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1141 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1201 tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     1261 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg
     1321 attcgaatcc cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
     1381 taagtaccgc ctatagagtc tataggccca caaaaaatgc tttcttcttt taatatactt
     1441 ttttgtttat cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
     1501 caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt ctgggttaag
     1561 gcaatagcaa tatttctgca tataaatatt tctgcatata aattgtaact gatgtaagag
     1621 gtttcatatt gctaatagca gctacaatcc agctaccatt ctgcttttat tttatggttg
     1681 ggataaggct ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct
     1741 cttatcttcc tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
     1801 ggcaaagaat tgggattcga acatcgattg aattcatggt gagcaagggc gaggaggata
     1861 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
     1921 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     1981 ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc
     2041 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     2101 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     2161 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     2221 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagaca atgggctggg
     2281 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
     2341 ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca
     2401 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc
     2461 acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg
     2521 gcggcatgga cgagctgtac aagtaatcta gagtcgacct gcagaagctt gcctcgagca
     2581 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     2641 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     2701 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     2761 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     2821 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     2881 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     2941 tgtttttttg gtagagacgg ggtttcacca tattggccag gctcgtctcc aactcctaat
     3001 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     3061 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     3121 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3181 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3241 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3301 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3361 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     3421 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3481 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3541 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3601 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3661 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     3721 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     3781 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3841 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3901 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3961 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4021 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4141 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4201 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4261 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4321 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4381 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4441 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4501 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4561 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4621 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4681 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4741 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4801 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4861 gctgataaat ctggagccgg tgagcgtggc tctcgcggta tcattgcagc actggggcca
     4921 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4981 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5041 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5101 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5161 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5221 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5281 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5341 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5401 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5461 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5521 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5581 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5641 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5701 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5761 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5821 ttcctggcct tttgctggcc ttttgctcac atgt
//
