LOCUS       sequence-464135-        6071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of 
            2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase 
            in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6071)
  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_1HKA
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     714..733
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     798..817
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      928..1516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1417..1436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1670..1687
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1702..1841
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1767..1786)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1927..1949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1943..2134)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2943..4025)
                     /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     3987..4006
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4026..4103)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4309..4328
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4416..4435
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4416..4434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4435..4459
                     /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             4474..4496
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4519..4548
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5347..5364
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5417..5435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5431..5478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5515..5970
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5602..5621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5812..5833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6062..6071,1..806))
                     /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(6062..6071,1..806))
                     /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 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
       61 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
      121 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
      181 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
      241 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
      301 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
      361 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
      421 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
      481 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
      541 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
      601 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
      661 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
      721 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
      781 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
      841 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
      901 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      961 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1021 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     1081 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1141 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1201 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1261 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1321 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1381 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1441 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1501 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1561 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1621 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1681 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     1741 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     1801 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     1861 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     1921 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     1981 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     2041 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     2101 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     2161 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     2221 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     2281 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     2341 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     2401 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     2461 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     2521 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     2581 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     2641 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     2701 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     2761 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     2821 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     2881 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     2941 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3001 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     3061 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     3121 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     3181 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc
     3241 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     3301 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     3361 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     3421 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     3481 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     3541 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     3601 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     3661 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     3721 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     3781 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     3841 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     3901 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     3961 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     4021 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     4081 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     4141 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     4201 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     4261 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     4321 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     4381 gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     4441 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     4501 catatgatgg cgaagattca tcaccatcat caccatcacc atcaccacgg tagcctgcaa
     4561 gattcggaag tcaaccaaga agcaaagccg gaagtcaagc cggaagtgaa accggaaacc
     4621 catattaacc tgaaagttag tgacggcagc tctgaaattt tctttaagat caaaaagacc
     4681 acgccgctgc gtcgcctgat ggaagcgttt gccaaacgtc agggcaagga aatggatagc
     4741 ctgcgtttcc tgtatgacgg tattcgcatc caggcagatc aagcgccgga agacctggac
     4801 atggaagaca acgacatcat tgaagcccac cgtgaacaga ttggcggctc tggaacagta
     4861 gcatatattg ctataggatc aaatttggcg tcgccgcttg agcaggtcaa tgctgcctta
     4921 aaagctttgg gcgatatccc ggagtctcat atcctgaccg ttagctcctt ttaccgcact
     4981 ccgccgttgg gcccacagga tcagccggat tacctgaatg cggcagttgc gttggaaacc
     5041 agcctggcgc ctgaggaact gctgaaccac acgcaacgta ttgagctgca gcaaggtcgt
     5101 gtgagaaagg cggaacgctg gggtccgcgt accttggacc tggacatcat gctgttcggt
     5161 aatgaagtta ttaacaccga acgcctgacg gtgccgcatt atgacatgaa gaaccgtggt
     5221 tttatgctgt ggccgctgtt cgagatcgcc ccggagctcg tgtttccgga cggcgaaatg
     5281 ttacgtcaaa ttctgcacac ccgtgcattc gataaactga acaaatgggg ttcgtaatga
     5341 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     5401 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     5461 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     5521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5581 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5701 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5761 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5821 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5881 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5941 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     6001 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     6061 cttagaaaaa c
//
