LOCUS       sequence-304401-        8309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for producing Avi tagged Alanyl-tRNA 
            synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8309)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 8309)
  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: AARS_C2_pNIC-Bio3
FEATURES             Location/Qualifiers
     source          1..8309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1455..1472
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1497..1517
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4440..4484
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4537..4554
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4607..4625)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4621..4668
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4705..5160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4792..4811)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5002..5023
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5252..6067)
                     /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(5252..6067)
                     /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     5975..5994
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6059..6078
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6189..6777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6678..6697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6931..6948
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6963..7102
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7028..7047)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7188..7210
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7204..7395)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8204..8309,1..977))
                     /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 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagctga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatataca tatgcaccat catcatcatc attcttctgg tgtagatctg ggtaccgaga
     1501 acctgtactt ccaatccatg gactctactc taacagcaag tgaaatccgg cagcgattta
     1561 tagatttctt caagaggaac gagcatacgt atgttcactc gtctgccacc atcccattgg
     1621 atgaccccac tttgctcttt gccaatgcag gcatgaacca gtttaaaccc attttcctga
     1681 acacaattga cccatctcac cccatggcaa agctgagcag agctgccaat acccagaagt
     1741 gcatccgggc tgggggcaaa cataatgacc tggacgatgt gggcaaggat gtctatcatc
     1801 acaccttctt cgagatgctg ggctcttggt cttttggaga ttactttaag gaattggcat
     1861 gtaagatggc tctggaactc ctcacccaag agtttggcat tcccattgaa agactttatg
     1921 ttacttactt tggcggggat gaagcagctg gcttagaagc agatctggaa tgcaaacaga
     1981 tctggcaaaa tttggggctg gatgacacca aaatcctccc aggcaacatg aaggataact
     2041 tctgggagat gggtgacacg ggcccctgtg gtccttgcag tgagatccac tacgaccgga
     2101 ttggtggtcg ggacgccgca catcttgtca accaggacga ccctaatgtg ctggagatct
     2161 ggaaccttgt gttcatccag tataacaggg aagctgatgg cattctgaaa cctcttccca
     2221 agaaaagcat tgacacaggg atgggcctgg aacgactggt atctgtgctg cagaataaga
     2281 tgtccaacta tgacactgac ctttttgtcc cttactttga agccattcag aagggcacag
     2341 gtgcccgacc atacactggg aaagttggtg ctgaggatgc cgatgggatt gacatggcct
     2401 accgggtgct ggctgaccat gctcggacca tcactgtggc actggctgat ggtggccggc
     2461 ctgacaacac agggcgtgga tatgtgttga gacggattct ccgccgagct gtccgatacg
     2521 cccatgaaaa gctcaatgcc agcaggggct tctttgctac gttagtggat gttgtcgtcc
     2581 agtccctggg agatgcattt cctgagctga agaaggaccc agacatggtg aaggacatca
     2641 ttaatgaaga agaggtgcag tttctcaaga ctctcagcag agggcgtcgc atcctggaca
     2701 ggaaaattca gagcctggga gacagcaaga ccattcccgg agacactgct tggctcctct
     2761 atgacaccta tgggtttcca gtggatctga ctggactgat tgctgaagag aagggcctgg
     2821 tggtagacat ggatggcttt gaagaggaga ggaaactggc ccagctgaaa tcacagggca
     2881 agggagctgg tggggaagac ctcattatgc tggacattta cgctatcgaa gagctccggg
     2941 cacggggtct ggaggtcaca gatgattccc caaagtacaa ttaccatttg gactccagtg
     3001 gtagctatgt atttgagaac acagtggcta cggtgatggc tctgcgcagg gagaagatgt
     3061 tcgtggaaga ggtgtccaca ggccaggagt gtggagtggt gctggacaag acctgtttct
     3121 atgctgagca aggaggccag atctatgacg aaggctacct ggtgaaggtg gatgacagca
     3181 gtgaagataa aacagagttt acagtgaaga atgctcaggt ccgaggaggg tatgtgctac
     3241 acattggaac catctacggt gacctgaaag tgggggatca ggtctggctg tttattgatg
     3301 agccccgacg aagacccatc atgagcaacc acacagctac gcacattctg aacttcgccc
     3361 tgcgctcagt gcttggggaa gctgaccaga aaggctcatt ggttgctcct gaccgcctca
     3421 gatttgactt tactgccaag ggagccatgt ccacccaaca gatcaagaag gctgaagaga
     3481 ttgctaatga gatgattgag gcagccaagg ccgtctatac ccaggattgc cccctggcag
     3541 cagcgaaagc catccagggc ctacgggctg tgtttgatga gacctatcct gaccctgtgc
     3601 gagtcgtctc cattggggtc ccggtgtccg agttgctgga tgacccctct gggcctgctg
     3661 gctccctgac ttctgttgag ttctgtgggg gaacgcacct gcggaactcg agtcatgcag
     3721 gagcttttgt gatcgtgacg gaagaagcca ttgccaaggg tatccggagg attgtggctg
     3781 tcacaggtgc cgaggcccag aaggccctca ggaaagcaga gagcttgaag aaatgtctct
     3841 ctgtcatgga agccaaagtg aaggctcaga ctgctccaaa caaggatgtg cagagggaga
     3901 tcgctgacct tggagaggcc ctggccactg cagtcatccc ccagtggcag aaggatgaat
     3961 tgcgggagac tctcaaatcc ctaaagaagg tcatggatga cttggaccga gccagcaaag
     4021 ccgatgtcca gaaacgagtg ttagagaaga cgaagcagtt catcgacagc aaccccaacc
     4081 agcctcttgt catcctggag atggagagcg gcgcctcagc caaggccctg aatgaagcct
     4141 tgaagctctt caagatgcac tcccctcaga cttctgccat gctcttcacg gtggacaatg
     4201 aggctggcaa gatcacgtgc ctgtgtcaag tcccccagaa tgcagccaat cggggcttaa
     4261 aagccagcga gtgggtgcag caggtgtcag gcttgatgga cggtaaaggt ggtggcaagg
     4321 atgtgtctgc acaggccaca ggcaagaacg ttggctgcct gcaggaggcg ctgcagctgg
     4381 ccacttcctt cgcccagctg cgcctcgggg atgtaaagaa cagcagtaaa ggtggatacg
     4441 gcctgaatga tatctttgaa gcgcagaaga ttgaatggca tgaatgataa ggatccgaat
     4501 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     4561 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     4621 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     4681 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4741 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4801 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4861 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4921 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4981 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5041 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5101 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5221 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     5281 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     5341 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     5401 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     5461 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     5521 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     5581 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     5641 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     5701 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     5761 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     5821 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     5881 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     5941 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     6001 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     6061 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     6121 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6181 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6241 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6301 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     6361 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6421 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6481 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6541 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6601 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6661 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6721 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6781 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6841 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6901 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6961 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     7021 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     7081 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     7141 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7201 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     7261 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     7321 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     7381 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     7441 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     7501 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     7561 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     7621 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     7681 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     7741 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     7801 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     7861 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     7921 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     7981 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     8041 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgtc
     8101 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     8161 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     8221 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     8281 tttgcgtatt gggcgccagg gtggttttt
//
