LOCUS       sequence-115882-        6664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses  Coumarinyl- tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6664)
  AUTHORS   Steinberg X, Galpin J, Nasir G, Sepulveda-Ugarte J, Sepulveda R, 
            Gonzalez-Nilo F, Islas LD, Ahern CA, Brauchi SE
  TITLE     A Rationally Designed Aminoacyl-tRNA Synthetase for Genetically 
            Encoded Fluorescent Amino Acids
  JOURNAL   bioRxiv, 10.1101/065227
REFERENCE   2  (bases 1 to 6664)
  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"; journalName: "bioRxiv, 
            10.1101/065227"
COMMENT     Sequence Label: pCDNA3.1_CoumRS
FEATURES             Location/Qualifiers
     source          1..6664
                     /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"
     CDS             2200..2223
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2258..2275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2264..2488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2534..2962
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2621..2640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2831..2852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2971..2991)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2976..3305
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3156..3291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3218..3237
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3372..4166
                     /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(3426..3445)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4036..4055
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4340..4461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4377..4396)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4431..4450
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4510..4526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4510..4526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4523..4545)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4534..4550
                     /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(4558..4564,4565..4582,4583..4588))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4603..4624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4741..4758)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4912..5497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4992..5011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5668..6459,6460..6528))
                     /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     6291..6310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6529..6633)
                     /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 gctggctagc
      901 gtttaaactt aagcttggta ccccaccatg gcaagcagta acttgattaa acaattgcaa
      961 gagcgggggc tggtagccca ggtgacggac gaggaagcgt tagcagagcg actggcgcaa
     1021 ggcccgatcg cgctcggttg cggcttcgat cctaccgctg acagcttgca tttggggcat
     1081 cttgttccat tggaatgcct gaaacgcttc cagcaggcgg gccacaagcc ggttgcgcac
     1141 gtaggcggcg cggggggtct gattggcgac ccgagcttca aagctgccga gcgtaagctg
     1201 aacaccgaag aaactgttca ggagtgggtg gacaaaatcc gtaagcaggt tgccccgttc
     1261 ctcgatttcg actgtggaga aaactatcat atcgcggcga acaactatga ctggttcggc
     1321 aatatgaatg tgctgacctt cctgcgcgat attggcaaac acttctccgt taaccagatg
     1381 atcaacaaag aagcggttaa gcagcgtctc aaccgtgaag atcaggggat ttcgttcact
     1441 gagttttcct acaacctgtt gcagggttat ggcatcgcct gtgggaacaa acagtacggt
     1501 gtggtgctgc aaattggtgg ttctgaccag tggggtaaca tcacttctgg tatcgacctg
     1561 acccgtcgtc tgcatcagaa tcaggtgttt ggcctgaccg ttccgctgat cactaaagca
     1621 gatggcacca aatttggtaa aactgaaggc ggcgcagtct ggttggatcc gaagaaaacc
     1681 agcccgtaca aattctacca gttctggatc aacactgcgg atgccgacgt ttaccgcttc
     1741 ctgaagttct tcacctttat gagcattgaa gagatcaacg ccctggaaga agaagataaa
     1801 aacagcggta aagcaccgcg cgcccagtat gtactggcgg agcaggtgac tcgtctggtt
     1861 cacggtgaag aaggtttaca ggcggcaaaa cgtattaccg aatgcctgtt cagcggttct
     1921 ttgagtgcgc tgagtgaagc ggacttcgaa cagctggcgc aggacggcgt accgatggtt
     1981 gagatggaaa agggcgcaga cctgatgcag gcactggtcg attctgaact gcaaccttcc
     2041 cgtggtcagg cacgtaaaac tatcgcctcc aatgccatca ccattaacgg tgaaaaacag
     2101 tccgatcctg aatacttctt taaagaagaa gatcgtctgt ttggtcgttt taccttactg
     2161 cgtcgcggta aaaagaatta ctgtctgatt tgctggaaag actacaaaga cgatgacgac
     2221 aagtaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2281 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2341 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2401 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2461 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2521 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2581 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2641 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2701 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2761 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2821 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2881 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2941 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3001 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3061 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3121 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3181 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3241 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3301 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3361 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3421 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3481 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3541 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3601 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3661 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3721 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3781 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3841 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3901 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3961 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4021 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4081 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4141 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4201 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4261 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4321 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4381 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4441 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4501 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4561 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4621 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4681 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4741 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4801 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4861 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4921 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4981 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5041 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5101 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5161 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5221 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5281 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5341 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5401 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg
     5461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5581 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5641 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5701 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5761 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5821 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5881 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5941 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6001 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6061 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6121 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6181 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6241 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6301 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6361 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6421 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6481 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6541 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6601 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6661 cgtc
//
