LOCUS       sequence-474533-        8426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human IARS2 with putative tRNA binding deficiency 
            mutations (K664A and K667A) in mammalian cells, also carrying 
            synonymous mutations described in Addgene #232340.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8426)
  TITLE     Mechanism of Isoleucyl-tRNA synthetase 2 regulating proliferation 
            and apoptosis of cervical cancer cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8426)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3.1-IARS2-K664A/K667A
FEATURES             Location/Qualifiers
     source          1..8426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1274..1282
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             2295..2306
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(4039..4056)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4045..4269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4315..4743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4402..4421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4612..4633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4752..4772)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4917..5050
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4977..4996
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5131..5925
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5185..5204)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5795..5814
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6099..6220
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6136..6155)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6190..6209
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6269..6285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6269..6285)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6282..6304)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6293..6309
                     /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)."
     promoter        complement(join(6317..6323,6324..6341,6342..6347))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6362..6383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6500..6517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6671..7259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6751..6770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7430..8221,8222..8290))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8053..8072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8291..8395)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgaccgga ccatgcgttg ggggctgcgc cctcgcgggc
      961 cgggcgcggc cgccctggcc actgcccgaa gtttgtgggg gacgccccgc cttccctgca
     1021 gcccgggatg gcaaggggcg acgaagaggc ttctggtgcg gtcggtctcc ggggccagta
     1081 accaccagcc gaactcgaat agtggcagat accgggacac ggtgctgctg ccgcagacga
     1141 gcttccccat gaagctgctg ggccgccagc agccggacac ggagctggag atccagcaga
     1201 aatgtggatt ttcagaactt tattcatggc aaagagaaag aaaagtaaag acagaatttt
     1261 gccttcatga tggacctcct tatgcaaacg gtgaccctca tgttggacat gctttaaata
     1321 agattttgaa agacatagcc aatcgattcc atatgatgaa tggctccaaa atacattttg
     1381 tgcccggctg ggattgtcat gggttgccca ttgaaataaa agtattatca gaacttggta
     1441 gagaagctca gaatctttca gctatggaaa ttagaaagaa agctagatca tttgctaaag
     1501 cagccattga gaaacagaaa tcagcattta ttcgttgggg aataatggca gattggaata
     1561 attgctacta tacatttgat gggaagtatg aagccaaaca gttgagaact ttttaccaaa
     1621 tgtatgataa gggcttggtt tatcgatctt acaaacctgt gttttggtct ccgtcatcta
     1681 ggactgcatt ggctgaagca gaacttgaat ataatcctga gcatgtcagt cgttcaatat
     1741 atgtaaaatt tcctctctta aagccttctc caaaattggc atctcttata gatggttcat
     1801 ctcctgttag tattttggtc tggaccacac aaccttggac gattccagcc aatgaagctg
     1861 tttgctatat gcctgaatca aagtatgctg ttgtgaaatg ttctaagtct ggagacctct
     1921 acgtactggc ggcagataaa gtagcatctg ttgcttctac tttggaaaca acatttgaga
     1981 ctatttcaac actttcaggt gtagatttgg aaaatggtac ttgcagccac cctctgattc
     2041 ctgataaagc ctctcctctt ttacctgcaa atcatgtgac catggcaaaa ggaacgggat
     2101 tggttcacac agccccagct catggtatgg aagactacgg tgtagcgtct cagcacaacc
     2161 tgcccatgga ttgtctagtg gacgaagatg gagttttcac agatgttgca ggtcctgaac
     2221 ttcaaaacaa ggctgtcctt gaagagggaa ctgatgtggt tataaagatg cttcagactg
     2281 caaagaattt gttgaaagag gagaaattgg tgcatagcta tccgtatgac tggaggacca
     2341 agaaacctgt ggttattcgt gccagcaagc agtggtttat aaacatcacg gatattaaga
     2401 ctgcagccaa ggaattgtta aaaaaggtga aatttattcc tggatcagca ctgaatggca
     2461 tggttgaaat gatggacagg cggccatatt ggtgtatatc aaggcaaaga gtttggggtg
     2521 ttccaattcc tgtgtttcat cataagacca aggatgaata cttgatcaac agccaaacca
     2581 ctgagcatat tgttaaacta gtggaacaac acggcagtga tatctggtgg actcttcccc
     2641 ctgaacaact tcttccaaaa gaagtcttat ctgaggttgg tggccctgat gccttggaat
     2701 atgtgccagg tcaggatatt ttggacatct ggtttgatag cggaacttca tggtcttatg
     2761 ttcttccagg tcctgaccaa agagcagatt tgtacttgga aggaaaagac cagctcgggg
     2821 gttggtttca gtcatcctta ttaacaagtg tggcagcaag gaagagagca ccttataaga
     2881 cagtgattgt tcatggattt acccttggag aaaagggaga agccatgtcc gcctctcttg
     2941 ggaatgtcat tcatcctgat gttgtcgtta atggaggaca agatcaaagc aaagagcctc
     3001 cgtatggtgc tgatgtcctt cgctggtggg tagctgattc caatgtcttc accgaagttg
     3061 caattggccc atccgtgctc aatgctgcca gagatgatat tagcaagctt aggaatacac
     3121 ttcgctttct tttgggaaat gtggctgatt tcaacccaga aacagattcc atccctgtaa
     3181 acgatatgta tgtcatagac cagtacatgc tacacttact gcaggatttg gcaaacaaga
     3241 ttaccgaatt atacaaacaa tatgattttg gaaaagttgt tcggctgtta cggacgtttt
     3301 ataccagaga gctctctaac ttttatttca gtataatcaa agataggctc tattgtgaaa
     3361 aggaaaatga ccccaaacga cgctcttgtc agactgcatt agttgaaatt ttggatgtaa
     3421 tagttcgttc ttttgctccc attcttcctc acctggctga agaggtgttc cagcacatac
     3481 cttatattaa agagcccaag agtgttttcc gtactgggtg gattagtact agttctatct
     3541 ggaaaaagcc cgggttggaa gaagctgtgg agagtgcgtg tgcaatgcga gactcatttc
     3601 ttggaagcat ccctggcaaa aatgcagctg agtacaaggt tatcactgtg atagaacctg
     3661 gactgctttt tgagataata gagatgctgc agtctgaaga gacttccagc acctctcagt
     3721 tgaatgaatt aatgatggct tctgagtcaa ctttactggc tcaggaacca cgagagatga
     3781 ctgcagatgt aatcgagctt aaagggaaat tcctcatcaa cttagaaggt ggtgatattc
     3841 gtgaagagtc ttcctataaa gtaattgtca tgccgactac gaaagaaaaa tgcccccgtt
     3901 gttggaagta tacagcggag tcttcagata cactgtgtcc tcgatgtgca gaagttgtca
     3961 gtggaaaata ggaattccac cacactggac tagtggatcc gagctcggta ccaagcttaa
     4021 gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     4081 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     4141 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     4201 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     4261 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     4321 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4381 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4441 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4501 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4561 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4621 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4681 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4741 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4801 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4861 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4921 gcccctaact ccgcccatcc cgcccattct ccgccccatg gctgactaat tttttttatt
     4981 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     5041 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     5101 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5161 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5221 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5281 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     5341 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5401 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5461 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5521 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5581 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5641 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5701 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5761 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5821 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5881 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5941 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     6001 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     6061 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     6121 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6181 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     6241 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     6301 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6361 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6421 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6481 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6541 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6601 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6661 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6721 gtcagaggtg gcgaaacccg gcaggactat aaagatacca ggcgtttccc cctggaagct
     6781 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6841 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6901 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6961 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7021 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7081 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7141 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7201 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7261 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7321 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7381 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7441 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7501 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7561 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7621 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7681 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7741 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7801 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7861 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7921 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7981 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8041 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8101 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8161 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8221 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8281 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8341 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8401 ttccccgaaa agtgccacct gacgtc
//
