LOCUS       sequence-83364-e        7580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an insect mitochondrial arginyl-tRNA synthetase in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7580)
  AUTHORS   Leisinger AK, Janzen DH, Hallwachs W, Igloi GL
  TITLE     Amino acid discrimination by the nuclear encoded mitochondrial 
            arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes 
            brasiliensis) from northwestern Costa Rica.
  JOURNAL   Insect Biochem Mol Biol. 2013 Dec;43(12):1172-80.
  PUBMED    24446543
REFERENCE   2  (bases 1 to 7580)
  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: "Insect 
            Biochem Mol Biol."; date: "2013-12"; volume: "43(12)"; pages: 
            "1172-80"
COMMENT     Sequence Label: pET-Cb2
FEATURES             Location/Qualifiers
     source          1..7580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1899..1913)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1929..1973)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(1980..1997)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2007..2024)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2046..2372)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             2380..2402
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2417..2441
                     /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     complement(2441..2460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2442..2460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2548..2567)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2773..2850
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2851..3933
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2870..2889)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4742..4933
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4927..4949)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5035..5177
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5090..5109
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5192..5209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5363..5951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5443..5462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6122..6913,6914..6982))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6745..6764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6983..7087)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7114..7569)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7251..7272)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7463..7482
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcctcacatt gcttgtaagg gctgaagacc tagtatttcc
      241 ataccattct tcaatacttc cctggcagtg ttaaaaagta atagtctttg ggatgctaca
      301 tctggactca tacctttgac ttgtagttca tggagtgcct tgttgatgta gttgcacaag
      361 tgaaacaggt agttcaccaa tatatacgct tctaactgcg catttgcctt atataacaca
      421 tcatgaaact ttgccagaac tttcagcaat accaacgctt cgggatcatc gaaaatctct
      481 ggattacatt ctttggcagg tgttgctcca gaattctttt ccaggctatg taagcggcag
      541 tgtgtgtact gaagccttat tccagtgtct ccctgaacct gaagtactgt atcccagttg
      601 aactcgtaat ccttttgtct tcgtcttttt aactcattaa taattacaca ggacacgccc
      661 aggatgtctg ctacttcacc atcattgata ggtaccttgg tagtaggtga ctcaacttgt
      721 cgttttatca tgatatccct gcattcatcc agaatatctt taagaaagat agatgttcct
      781 tttcttgtac tcatgcccct tatcctacca aacttgacat gaactaatct ccctgcccaa
      841 ggtaagtcca ttttatatag aatatcctta agtgtataaa gatgatcact ctggctgttg
      901 tcaacaacat aatacatctt atcaaaactg tattttttga atctatctat tgctgcagct
      961 atatcgcgag ttagatataa ggtagaacca tcactcttca gaactgacac ctttttttca
     1021 tgtatagtag caacttcttt tccatcatta cttttctgta taatgttctt agttctcaac
     1081 atttcaataa catttccaat gttctttgca ctatagtctg attcataact atagtcgtca
     1141 aatgatattc ctaacctgtt ataagtattg atcaattctt ttttagagta gtccatgtaa
     1201 gtttgccaat cttttatatc atcaactgaa cctatttcta actttaagaa ttcttgcttc
     1261 gctctgttta gtatactgga atctttctca gctaatttgt tagcatggac atatgattta
     1321 tacagcagtg tcaaagggtc tttttttata tcttctggtg tatatttgag ttcttcaata
     1381 ccaactttta tgaggccaaa ctgtgtcccc caatttccca cataatttaa tcttactact
     1441 ttgtgcctta aaaaactatt gaggttgctt ataaaatttc ctataatagt agacctgagg
     1501 tgtcctaagt gaaatggttt agcaacattt ggtgaactga agtcaacaac aatatttttt
     1561 gggctgaaat ttaactctgg aaatgcacag ttttcaatca catctttgat aaatatagtt
     1621 ttatctattt caaatgatat cctcctgttt gcgcgatctc ttacgaggta aatgtttttt
     1681 atgatatcat caggctgaat ctttaaaacc tcggttatgt tcgttacacc taactgcctt
     1741 tccaaatcgt ttaaaggtaa actcatggaa attttactct catctgcatt tcctatttga
     1801 atcagcggtt gaagctctgc taacgaaaat tttgtgtatt tgtgtagaca gtcgatcacc
     1861 ttccttccta ggtacaattt tagtttagtt gccatggcct tgtcgtcgtc gtcggtaccc
     1921 agatctgggc tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatacca
     1981 gaaccgcgtg gcaccagacc agaagaatga tgatgatgat ggtgcatatg gccagaacca
     2041 gaaccggcca ggttagcgtc gaggaactct ttcaactgac ctttagacag tgcacccact
     2101 ttggttgccg ccacttcacc gtttttgaac agcagcagag tcgggatacc acggatgcca
     2161 tatttcggcg cagtgccagg gttttgatcg atgttcagtt ttgcaacggt cagtttgccc
     2221 tgatattcgt cagcgatttc atccagaatc ggggcgatca ttttgcacgg accgcaccac
     2281 tctgcccaga aatcgacgag gatcgccccg tccgctttga gtacatccgt gtcaaaactg
     2341 tcgtcagtca ggtgaataat tttatcgctc atatgtatat ctccttctta aagttaaaca
     2401 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
     2461 atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     2521 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     2581 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     2641 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     2701 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     2761 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     2821 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     2881 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     2941 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     3001 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     3061 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     3121 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     3181 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     3241 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     3301 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     3361 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     3421 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     3481 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     3541 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     3601 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga catctcggta
     3661 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     3721 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     3781 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     3841 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3901 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     3961 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     4021 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     4081 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     4141 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     4201 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     4261 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     4321 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     4381 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     4441 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     4501 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     4561 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     4621 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     4681 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     4741 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     4801 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     4861 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     4921 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4981 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     5041 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     5101 cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga
     5161 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5221 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5281 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5341 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5401 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5461 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5521 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5581 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5641 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5701 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5761 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5821 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5881 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5941 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6001 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6061 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6121 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6181 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6241 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6301 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6361 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6421 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6481 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6541 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6601 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6661 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6721 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6781 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6841 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6901 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6961 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7021 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7081 ttccgcgcac atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa
     7141 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     7201 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     7261 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     7321 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     7381 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     7441 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     7501 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     7561 agggcgcgtc ccattcgcca
//
