LOCUS       sequence-304463-        7460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for producing Avi tagged Aspartyl-tRNA 
            synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7460)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 7460)
  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"
COMMENT     Sequence Label: GARS_pNIC-Bio3
FEATURES             Location/Qualifiers
     source          1..7460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    186..325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(251..270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     411..433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(427..618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1427..2509)
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2471..2490
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2510..2587)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2793..2812
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2896..2915
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2896..2914
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2915..2939
                     /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)."
     RBS             2954..2976
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2987..3004
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3029..3049
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(3888..3899)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5123..5167
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5220..5237
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5290..5308)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5304..5351
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5388..5843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5475..5494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5685..5706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5935..6750)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5935..6750)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6658..6677
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6742..6761
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6872..7460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7361..7380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
      241 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      301 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      361 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      421 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      481 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      541 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      601 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      661 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      721 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      781 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      841 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
      901 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      961 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     1021 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     1081 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1141 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1201 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1261 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     1321 gtcccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     1381 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     1441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1501 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     1561 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     1621 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     1681 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     1741 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     1801 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     1861 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     1921 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     1981 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     2041 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     2101 ccacagcaat ggcatcctgg tcatccagct gatagttaat gatcagccca ctgacgcgtt
     2161 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2221 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     2281 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     2341 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     2401 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     2461 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     2521 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     2581 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     2641 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     2701 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     2761 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     2821 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     2881 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     2941 ctctagaaat aattttgttt aactttaaga aggagatata catatgcacc atcatcatca
     3001 tcattcttct ggtgtagatc tgggtaccga gaacctgtac ttccaatcca tggacggcgc
     3061 gggggctgag gaggtgctgg cacctctgag gctagcagtg cgccagcagg gagatcttgt
     3121 gcgaaaactc aaagaagata aagcacccca agtagacgta gacaaagcag tggctgagct
     3181 caaagcccgc aagagggttc tggaagcaaa ggagctggcg ttacagccca aagatgatat
     3241 tgtagaccga gcaaaaatgg aagataccct gaagaggagg tttttctatg atcaagcttt
     3301 tgctatttat ggaggtgtta gtggtctgta tgactttggg ccagttggct gtgctttgaa
     3361 gaacaatatt attcagacct ggaggcagca ctttatccaa gaggaacaga tcctggagat
     3421 cgattgcacc atgctcaccc ctgagccagt tttaaagacc tctggccatg tagacaaatt
     3481 tgctgacttc atggtgaaag acgtaaaaaa tggagaatgt tttcgtgctg accatctatt
     3541 aaaagctcat ttacagaaat tgatgtctga taagaagtgt tctgtcgaaa agaaatcaga
     3601 aatggaaagt gttttggccc agcttgataa ctatggacag caagaacttg cggatctttt
     3661 tgtgaactat aatgtaaaat ctcccattac tggaaatgat ctatcccctc cagtgtcttt
     3721 taacttaatg ttcaagactt tcattgggcc tggaggaaac atgcctgggt acttgagacc
     3781 agaaactgca caggggattt tcttgaattt caaacgactt ttggagttca accaaggaaa
     3841 gttgcctttt gctgctgccc agattggaaa ttcttttaga aatgagatct cccctcgatc
     3901 tggactgatc agagtcagag aattcacaat ggcagaaatt gagcactttg tagatcccag
     3961 tgagaaagac caccccaagt tccagaatgt ggcagacctt cacctttatt tgtattcagc
     4021 aaaagcccag gtcagcggac agtccgctcg gaaaatgcgc ctgggagatg ctgttgaaca
     4081 gggtgtgatt aataacacag tattaggcta tttcattggc cgcatctacc tctacctcac
     4141 gaaggttgga atatctccag ataaactccg cttccggcag cacatggaga atgagatggc
     4201 ccattatgcc tgtgactgtt gggatgcaga atccaaaaca tcctacggtt ggattgagat
     4261 tgttggatgt gctgatcgtt cctgttatga cctctcctgt catgcacgag ccaccaaagt
     4321 cccacttgta gctgagaaac ctctgaaaga acccaaaaca gtcaatgttg ttcagtttga
     4381 acccagtaag ggagcaattg gtaaggcata taagaaggat gcaaaactgg tgatggagta
     4441 tcttgccatt tgtgatgagt gctacattac agaaatggag atgctgctga atgagaaagg
     4501 ggaattcaca attgaaactg aagggaaaac atttcagtta acaaaagaca tgatcaatgt
     4561 gaagagattc cagaaaacac tatatgtgga agaagttgtt ccgaatgtaa ttgaaccttc
     4621 cttcggcctg ggtaggatca tgtatacggt atttgaacat acattccatg tacgagaagg
     4681 agatgaacag agaacattct tcagtttccc tgctgtagtt gctccattca aatgttccgt
     4741 cctcccactg agccaaaacc aggagttcat gccatttgtc aaggaattat cggaagccct
     4801 gaccaggcat ggagtatctc acaaagtaga cgattcctct gggtcaatcg gaaggcgcta
     4861 tgccaggact gatgagattg gcgtggcttt tggtgtcacc attgactttg acacagtgaa
     4921 caagaccccc cacactgcaa ctctgaggga ccgtgactca atgcggcaga taagagcaga
     4981 gatctctgag ctgcccagca tagtccaaga cctagccaat ggcaacatca catgggctga
     5041 tgtggaggcc aggtatcctc tgtttgaagg gcaagagact ggtaaaaaag agacaatcga
     5101 ggaaagcagt aaaggtggat acggcctgaa tgatatcttt gaagcgcaga agattgaatg
     5161 gcatgaatga taaggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc
     5221 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5281 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5341 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     5401 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5461 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5521 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5581 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5641 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5701 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5761 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5821 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5881 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     5941 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     6001 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     6061 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     6121 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     6181 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     6241 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     6301 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     6361 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     6421 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     6481 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6541 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     6601 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     6661 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     6721 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     6781 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6841 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6901 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6961 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     7021 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7081 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7141 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7201 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7261 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7321 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7381 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7441 gggggcggag cctatggaaa
//
