LOCUS       sequence-304477-        6620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for producing Avi tagged Threonyl-tRNA 
            synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 6620)
  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: TARS_pNIC-Bio3
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1486..1503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1528..1548
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2782..2826
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2879..2896
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2949..2967)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2963..3010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3047..3502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3134..3153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3344..3365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3594..4409)
                     /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(3594..4409)
                     /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     4317..4336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4401..4420
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4531..5119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5020..5039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5273..5290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5305..5444
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5370..5389)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5530..5552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5546..5737)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6546..6620,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagctg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat
     1501 cattcttctg gtgtagatct gggtaccgag aacctgtact tccaatccat gaaccgagat
     1561 cataggaaaa ttggcaggga ccaagaacta tatttctttc atgaactcag ccctggaagt
     1621 tgcttttttc tgccaaaagg agcctacatt tataatgcac ttattgaatt cattaggagc
     1681 gaatatagga aaagaggatt ccaggaggta gtcaccccaa acatcttcaa cagccgactc
     1741 tggatgacct cgggccactg gcagcactac agcgagaaca tgttctcctt tgaggtggag
     1801 aaggagctgt ttgccctgaa acccatgaac tgcccaggac actgccttat gtttgatcat
     1861 cggccaaggt cctggcgaga actgcctctg cggctagctg attttggggt acttcatagg
     1921 aacgagctgt ctggagcact cacaggactc acccgggtac gaagattcca acaggatgat
     1981 gctcacatat tctgtgccat ggagcagatt gaagatgaaa taaaaggttg tttggatttt
     2041 ctacgtacgg tatatagcgt atttggattt tcttttaaac taaacctttc tactcgcccg
     2101 gaaaaattcc ttggagatat cgaagtatgg gatcaagctg agaaacaact tgaaaacagt
     2161 ctgaatgaat ttggtgaaaa gtgggagtta aactctggag atggagcttt ctatggccca
     2221 aagattgaca tacagattaa agatgcgatt gggcggtacc accagtgtgc aaccatccag
     2281 ctggatttcc agttgcccat cagatttaat cttacttatg taagccatga tggtgatgat
     2341 aagaaaaggc cagtgattgt tcatcgagcc atcttgggat cagtggaaag aatgattgct
     2401 atcctcacag aaaactatgg gggcaaatgg cccttttggc tgtcccctcg ccaggtaatg
     2461 gtagttccag tgggaccaac ctgtgatgaa tatgcccaaa aggtacgaca acaattccac
     2521 gatgccaaat tcatggcaga cattgatctg gatccaggct gtacattgaa taaaaagatt
     2581 cgaaatgcac agttagcaca gtataacttc attttagttg ttggtgaaaa agagaaaatc
     2641 agtggcactg ttaatatccg cacaagagac aataaggtcc acggggaacg caccatttct
     2701 gaaactatcg agcggctaca gcagctcaaa gagttccgca gcaaacaggc agaagaagaa
     2761 tttagcagta aaggtggata cggcctgaat gatatctttg aagcgcagaa gattgaatgg
     2821 catgaatgat aaggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca
     2881 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2941 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     3001 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     3061 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3301 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3361 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3421 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3481 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3541 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     3601 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3661 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3721 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3781 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3841 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3901 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3961 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4021 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4081 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     4141 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4201 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4261 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4321 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     4381 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4441 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4501 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4561 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4621 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4681 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4741 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4801 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4861 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4921 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4981 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5041 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5101 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5161 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5221 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5281 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5341 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5401 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5461 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5521 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5581 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5641 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5701 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5761 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5821 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5881 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5941 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     6001 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     6061 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     6121 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     6181 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     6241 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     6301 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     6361 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6421 gtgcggcgac gatagtcatg tcccgcgccc accggaagga gctgactggg ttgaaggctc
     6481 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6541 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6601 gccaacgcgc ggggagaggc
//
