LOCUS       sequence-304466-        7196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for producing Avi tagged Lysyl-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7196)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 7196)
  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: KARS_pNIC-Bio3
FEATURES             Location/Qualifiers
     source          1..7196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(79..1161)
                     /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     1123..1142
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1162..1239)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1445..1464
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1548..1567
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1548..1566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1567..1591
                     /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             1606..1628
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1639..1656
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1681..1701
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      1700..1709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3511..3555
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             3608..3625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3678..3696)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3692..3739
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3776..4231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3863..3882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4073..4094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4323..5138)
                     /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(4323..5138)
                     /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     5046..5065
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5130..5149
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5260..5848
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5749..5768
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6002..6019
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6034..6173
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6099..6118)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6259..6281
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6275..6466)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
       61 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
      181 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
      241 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
      301 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
      361 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
      421 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
      481 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
      541 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
      601 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
      661 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
      721 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
      781 ctgatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
      841 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
      901 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
      961 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     1021 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     1081 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     1141 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     1201 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     1261 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     1321 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     1381 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     1441 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     1501 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     1561 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     1621 gaaggagata tacatatgca ccatcatcat catcattctt ctggtgtaga tctgggtacc
     1681 gagaacctgt acttccaatc catggcggcc gtgcaggcgg ccgaggtgaa agtggatggc
     1741 agcgagccga aactgagcaa gaatgagctg aagagacgcc tgaaagctga gaagaaagta
     1801 gcagagaagg aggccaaaca gaaggagctc agtgagaaac agctaagcca agccactgct
     1861 gctgccacca accacaccac tgataatggt gtgggtcctg aggaagagag cgtggaccca
     1921 aatcaatact acaaaatccg cagtcaagca attcatcagc tgaaggtcaa tggggaagac
     1981 ccatacccac acaagttcca tgtagacatc tcactcactg acttcatcca aaaatatagt
     2041 cacctgcagc ctggggatca cctgactgac atcaccttaa aggtggcagg taggatccat
     2101 gccaaaagag cttctggggg aaagctcatc ttctatgatc ttcgaggaga gggggtgaag
     2161 ttgcaagtca tggccaattc cagaaattat aaatcagaag aagaatttat tcatattaat
     2221 aacaaactgc gtcggggaga cataattgga gttcagggga atcctggtaa aaccaagaag
     2281 ggtgagctga gcatcattcc gtatgagatc acactgctgt ctccctgttt gcatatgtta
     2341 cctcatcttc actttggcct caaagacaag gaaacaaggt atcgccagag atacttggac
     2401 ttgatcctga atgactttgt gaggcagaaa tttatcatcc gctctaagat catcacatat
     2461 ataagaagtt tcttagatga gctgggattc ctagagattg aaactcccat gatgaacatc
     2521 atcccagggg gagccgtggc caagcctttc atcacttatc acaacgagct ggacatgaac
     2581 ttatatatga gaattgctcc agaactctat cataagatgc ttgtggttgg tggcatcgac
     2641 cgggtttatg aaattggacg ccagttccgg aatgagggga ttgatttgac gcacaatcct
     2701 gagttcacca cctgtgagtt ctacatggcc tatgcagact atcacgatct catggaaatc
     2761 acggagaaga tggtttcagg gatggtgaag catattacag gcagttacaa ggtcacctac
     2821 cacccagatg gcccagaggg ccaagcctac gatgttgact tcaccccacc cttccggcga
     2881 atcaacatgg tagaagagct tgagaaagcc ctggggatga agctgccaga aacgaacctc
     2941 tttgaaactg aagaaactcg caaaattctt gatgatatct gtgtggcaaa agctgttgaa
     3001 tgccctccac ctcggaccac agccaggctc cttgacaagc ttgttgggga gttcctggaa
     3061 gtgacttgca tcaatcctac attcatctgt gatcacccac agataatgag ccctttggct
     3121 aaatggcacc gctctaaaga gggtctgact gagcgctttg agctgtttgt catgaagaaa
     3181 gagatatgca atgcgtatac tgagctgaat gatcccatgc ggcagcggca gctttttgaa
     3241 gaacaggcca aggccaaggc tgcaggtgat gatgaggcca tgttcataga tgaaaacttc
     3301 tgtactgccc tggaatatgg gctgcccccc acagctggct ggggcatggg cattgatcga
     3361 gtcgccatgt ttctcacgga ctccaacaac atcaaggaag tacttctgtt tcctgccatg
     3421 aaacccgaag acaagaagga gaatgtagca accactgata cactggaaag cacaacagtt
     3481 ggcacttctg tcagcagtaa aggtggatac ggcctgaatg atatctttga agcgcagaag
     3541 attgaatggc atgaatgata aggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc
     3601 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     3661 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3721 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3781 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3841 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3901 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3961 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4021 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4081 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4141 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4201 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     4261 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     4321 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     4381 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     4441 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     4501 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     4561 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     4621 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     4681 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     4741 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     4801 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     4861 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4921 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4981 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     5041 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     5101 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     5161 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     5221 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5281 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5341 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5401 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5461 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5521 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5581 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5641 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5701 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5761 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5821 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5881 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5941 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6001 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     6061 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     6121 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     6181 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     6241 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     6301 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     6361 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     6421 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     6481 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     6541 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     6601 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     6661 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     6721 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     6781 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     6841 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     6901 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     6961 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     7021 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     7081 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     7141 cagtcataag tgcggcgacg atagtcatgt cccgcgccca ccggaaggag ctgact
//
