LOCUS       sequence-304465-        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for producing Avi tagged histidyl tRNA 
            synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 6932)
  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: HARS_pNIC-Bio3
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(40..1122)
                     /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     1084..1103
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1123..1200)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1406..1425
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1509..1528
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1509..1527
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1528..1552
                     /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             1567..1589
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1600..1617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1642..1662
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3208..3252
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             3305..3322
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3375..3393)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3389..3436
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3473..3928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3560..3579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3770..3791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4020..4835)
                     /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(4020..4835)
                     /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     4743..4762
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4827..4846
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4957..5545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5446..5465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5699..5716
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5731..5870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5796..5815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5956..5978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5972..6163)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
       61 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      121 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
      181 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      241 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      301 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      361 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      421 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      481 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      541 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      601 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      661 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      721 aatggcatcc tggtcatcca gctgatagtt aatgatcagc ccactgacgc gttgcgcgag
      781 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      841 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      901 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      961 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     1021 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     1081 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1141 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1201 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1261 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     1321 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     1381 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     1441 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     1501 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1561 aataattttg tttaacttta agaaggagat atacatatgc accatcatca tcatcattct
     1621 tctggtgtag atctgggtac cgagaacctg tacttccaat ccatggcaga gcgtgcggcg
     1681 ctggaggagc tggtgaaact tcagggagag cgcgtgcgag gcctcaagca gcagaaggcc
     1741 agcgccgagc tgatcgagga ggaggtggcg aaactcctga aactgaaggc acagctgggt
     1801 cctgatgaaa gcaaacagaa atttgtgctc aaaaccccca agggcacaag agactatagt
     1861 ccccggcaga tggcagttcg cgagaaggtg tttgacgtaa tcatccgttg cttcaagcgc
     1921 cacggtgcag aagtcattga tacacctgta tttgaactaa aggaaacact gatgggaaag
     1981 tatggggaag actccaagct tatctatgac ctgaaggacc agggcgggga gctcctgtcc
     2041 cttcgctatg acctcactgt tccttttgct cggtatttgg caatgaataa actgaccaac
     2101 attaaacgct accacatagc aaaggtatat cggcgggata acccagccat gacccgtggc
     2161 cgataccggg aattctacca gtgtgatttt gacattgctg ggaactttga tcccatgatc
     2221 cctgatgcag agtgcctgaa gatcatgtgc gagatcctga gttcacttca gataggcgac
     2281 ttcctggtca aggtaaacga tcgacgcatt ctagatggga tgtttgctat ctgtggtgtt
     2341 tctgacagca agttccgtac catctgctcc tcagtagaca agctggacaa ggtgtcctgg
     2401 gaagaggtga agaatgagat ggtgggagag aagggccttg cacctgaggt ggctgaccgc
     2461 attggggact atgtccagca acatggtggg gtatccctgg tggaacagct gctccaggat
     2521 cctaaactat cccaaaacaa gcaggccttg gagggcctgg gagacctgaa gttgctcttt
     2581 gagtacctga ccctatttgg cattgatgac aaaatctcct ttgacctgag ccttgctcga
     2641 gggctggatt actacactgg ggtgatctat gaggcagtgc tgctacagac cccagcccag
     2701 gcaggggaag agcccctggg tgtgggcagt gtggctgctg gaggacgcta tgatgggcta
     2761 gtgggcatgt tcgaccccaa agggcgcaag gtgccatgtg tggggctcag cattggggtg
     2821 gagcggattt tctccatcgt ggaacagaga ctagaggctt tggaggagaa gatacggacc
     2881 acggagacac aggtgcttgt ggcatctgca cagaagaagc tgctagagga aagactaaag
     2941 cttgtctcag aactgtggga tgctgggatc aaggctgagc tgctgtacaa gaagaaccca
     3001 aagctactga accagttaca gtactgtgag gaggcaggca tcccactggt ggctatcatc
     3061 ggcgagcagg aactcaagga tggggtcatc aagctccgtt cagtgacgag cagggaagag
     3121 gtggatgtcc gaagagaaga ccttgtggag gaaatcaaaa ggagaacagg ccagcccctc
     3181 tgcatctgca gcagtaaagg tggatacggc ctgaatgata tctttgaagc gcagaagatt
     3241 gaatggcatg aatgataagg atccgaattc gagctccgtc gacaagcttg cggccgcact
     3301 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     3361 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3421 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     3481 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3541 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3601 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3661 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3721 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3781 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3841 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3901 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     4021 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     4081 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     4141 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     4201 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     4261 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     4321 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     4381 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     4441 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     4501 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     4561 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     4621 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     4681 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     4741 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     4801 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     4861 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4921 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4981 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5041 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5101 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5161 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5221 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5281 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5341 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5401 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5461 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5521 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5581 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5641 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5701 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5761 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5821 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     5881 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5941 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6001 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     6061 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     6121 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     6181 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     6241 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     6301 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     6361 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     6421 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     6481 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     6541 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     6601 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     6661 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     6721 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     6781 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     6841 tcataagtgc ggcgacgata gtcatgtccc gcgcccaccg gaaggagctg actgggttga
     6901 aggctctcaa gggcatcggt cgagatcccg gt
//
