LOCUS       sequence-464171-        6041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of ribonuclease HI (RNase HI) in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6041)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6041)
  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_1JL1
FEATURES             Location/Qualifiers
     source          1..6041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     567..586
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     651..670
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      781..1369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1270..1289
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1523..1540
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1555..1694
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1620..1639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1780..1802
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1796..1987)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2796..3878)
                     /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     3840..3859
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3879..3956)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4162..4181
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4269..4288
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4269..4287
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4288..4312
                     /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             4327..4349
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4372..4401
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5170..5187
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5240..5258)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5254..5301
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5338..5793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5425..5444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5635..5656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5885..6041,1..659))
                     /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(5885..6041,1..659))
                     /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 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
       61 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
      121 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
      181 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
      241 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
      301 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
      361 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
      421 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
      481 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
      541 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
      601 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
      661 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
      721 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      781 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      841 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      901 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      961 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1021 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1081 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1141 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1201 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1261 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1321 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1381 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1441 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     1501 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     1561 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     1621 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     1681 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     1741 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1801 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1861 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1921 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1981 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2041 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2101 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2161 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     2221 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     2281 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     2341 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     2401 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     2461 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     2521 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     2581 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     2641 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     2701 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     2761 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2821 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2881 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2941 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3001 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     3061 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     3121 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     3181 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     3241 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     3301 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     3361 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     3421 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     3481 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     3541 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     3601 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     3661 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     3721 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3781 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3841 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3901 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3961 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4021 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     4081 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     4141 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     4201 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     4261 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     4321 aataattttg tttaacttta agaaggagat atacatatga tggcgaagat tcatcaccat
     4381 catcaccatc accatcacca cggtagcctg caagattcgg aagtcaacca agaagcaaag
     4441 ccggaagtca agccggaagt gaaaccggaa acccatatta acctgaaagt tagtgacggc
     4501 agctctgaaa ttttctttaa gatcaaaaag accacgccgc tgcgtcgcct gatggaagcg
     4561 tttgccaaac gtcagggcaa ggaaatggat agcctgcgtt tcctgtatga cggtattcgc
     4621 atccaggcag atcaagcgcc ggaagacctg gacatggaag acaacgacat cattgaagcc
     4681 caccgtgaac agattggcgg ctctggacaa gtagaaatat ttacagctgg atcagcatta
     4741 ggtaatccgg gtccgggcgg ttacggcgca atcctgcgtt atagaggccg tgaaaaaacg
     4801 ttcagcgcag gctacacccg taccaccaac aatcgtatgg aactgatggc tgcgatcgtg
     4861 gcgttggagg cgctgaaaga gcacgccgaa gttattctct ccaccgacag ccaatatgtg
     4921 cgccaaggta ttacccagtg gatccacaac tggaagaagc gcggttggaa gaccgctgac
     4981 aaaaaaccgg ttaagaacgt cgatctgtgg caacgtctgg atgcggcctt gggccagcat
     5041 cagattaaat gggagtgggt gaagggccac gcgggtcatc cggagaacga gcgcgcggac
     5101 gaactggcac gtgcggcggc tatgaaccca acgctggaag atactggtta cggttcgtaa
     5161 tgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     5221 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     5281 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     5341 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5401 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5461 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5521 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5581 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5641 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5701 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5761 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     5881 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     5941 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     6001 tccataggat ggcaagatcc tggtatcggt ctgcgattcc g
//
