LOCUS       sequence-464168-        6269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of threonine--tRNA ligase (EC 
            6.1.1.3) (Threonyl-tRNA synthetase) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6269)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6269)
  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: pAIP4_1TJE
FEATURES             Location/Qualifiers
     source          1..6269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     668..687
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     752..771
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      882..1470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1371..1390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1624..1641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1656..1795
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1721..1740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1881..1903
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1897..2088)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2897..3979)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3941..3960
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3980..4057)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4263..4282
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4370..4389
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4370..4388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4389..4413
                     /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             4428..4450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4473..4502
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5499..5516
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5569..5587)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5583..5630
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5667..6122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5754..5773)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5964..5985
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6214..6269,1..760))
                     /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(join(6214..6269,1..760))
                     /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"
ORIGIN
        1 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
       61 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
      121 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
      181 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
      241 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
      301 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
      361 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
      421 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
      481 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
      541 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
      601 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
      661 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
      721 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
      781 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
      841 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      901 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      961 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1021 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1081 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1141 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1201 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1261 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1321 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1381 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1441 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1501 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1561 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1621 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     1681 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     1741 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     1801 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     1861 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     1921 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     1981 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     2041 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     2101 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     2161 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     2221 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     2281 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     2341 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     2401 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     2461 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     2521 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     2581 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     2641 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     2701 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     2761 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     2821 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     2881 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2941 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3001 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3061 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3121 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     3181 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     3241 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     3301 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     3361 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3421 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3481 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     3541 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     3601 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     3661 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3721 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3781 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3841 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3901 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3961 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4021 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     4081 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     4141 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     4201 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     4261 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     4321 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt aatacgactc
     4381 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     4441 aagaaggaga tatacatatg atggcgaaga ttcatcacca tcatcaccat caccatcacc
     4501 acggtagcct gcaagattcg gaagtcaacc aagaagcaaa gccggaagtc aagccggaag
     4561 tgaaaccgga aacccatatt aacctgaaag ttagtgacgg cagctctgaa attttcttta
     4621 agatcaaaaa gaccacgccg ctgcgtcgcc tgatggaagc gtttgccaaa cgtcagggca
     4681 aggaaatgga tagcctgcgt ttcctgtatg acggtattcg catccaggca gatcaagcgc
     4741 cggaagacct ggacatggaa gacaacgaca tcattgaagc ccaccgtgaa cagattggcg
     4801 gctctggaat gccagtaata acactacccg atggatcaca acgccactac gaccacgccg
     4861 ttagcccgat ggatgtggcg ctggacatcg gtccgggtct ggctaaagcg tgcattgctg
     4921 gtcgtgttaa tggcgaattg gttgacgcct gcgatcttat cgaaaatgat gcgcagctgt
     4981 cgattatcac cgcgaaagat gaagaaggtt tggagatcat ccgccatagc tgtgcacacc
     5041 tgctgggtca tgcgattaag cagttatggc cgcacaccaa gatggctatc ggcccagtca
     5101 tcgacaacgg cttttattac gacgtcgacc tggaccgtac cttgacgcaa gaagatgttg
     5161 aagccttgga gaagcgcatg cacgagctgg cggaaaagaa ctatgacgtg atcaagaaga
     5221 aggtgagctg gcatgaggcg cgtgaaacct ttgcaaatag aggcgagagc tacaaagtgt
     5281 ccattctgga tgagaacatt gcgcacgatg acaaaccggg tctgtacttc cacgaggagt
     5341 atgtggacat gtgtcgtggt ccgcatgttc cgaacatgcg tttctgccat catttcaaac
     5401 tgatgaaaac cgctggcgca tattggcgtg gtgattctaa caacaaaatg ctgcagcgta
     5461 tttacggcac tgcgtgggca ggttcgtaat gactcgagca ccaccaccac caccactgag
     5521 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     5581 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     5641 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     5701 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5761 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5821 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5881 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5941 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     6001 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     6061 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     6121 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6181 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     6241 aaactgcaat ttattcatat caggattat
//
