LOCUS       sequence-464152-        6005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of thermonuclease (TNase) in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6005)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6005)
  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_4QB4
FEATURES             Location/Qualifiers
     source          1..6005
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(66..87)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     278..297
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(421..468)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     464..482
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(535..552)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1285..1314)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1337..1359
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1374..1398
                     /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)."
     primer_bind     complement(1398..1417)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1399..1417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1505..1524)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1730..1807
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1808..2890
                     /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     complement(1827..1846)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3699..3890
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3884..3906)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3992..4131
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4047..4066
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4146..4163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4317..4905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4397..4416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5016..5035)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5027..5842
                     /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             5027..5842
                     /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     complement(5100..5119)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5934..6005,1..384))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
       61 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
      121 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      181 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      241 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      301 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      361 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
      421 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
      481 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg
      541 gtggtggtgg tgctcgagtc attacgaacc ggaccagatg ttcagcttct ctttctttgc
      601 ctgcgcttcg cttttacgca ggtgctgttc atgggtgttg ttcggtttat aaacgtaagc
      661 aactttggct aaaccctggc gcaccagcgc ttcattaacc attttgccgt cagcataaat
      721 gtacgccaaa ccacggccgt atttatcggt acgttggccc ttgtcgaact cgacctcgat
      781 cttctttgca ttttccagca tctttttggt gaacgcgctg gcctccggac cgtacttctc
      841 tacacccttt tttgggtgct tggtttccgg agtgtccacg agcaacaggc gaaaggtcat
      901 cggttggcct ttatacatca ctttcagcgt atcaccatca atcgccttta ttagtgtagc
      961 gggttctttg tgtaactttc cagagccgcc aatctgttca cggtgggctt caatgatgtc
     1021 gttgtcttcc atgtccaggt cttccggcgc ttgatctgcc tggatgcgaa taccgtcata
     1081 caggaaacgc aggctatcca tttccttgcc ctgacgtttg gcaaacgctt ccatcaggcg
     1141 acgcagcggc gtggtctttt tgatcttaaa gaaaatttca gagctgccgt cactaacttt
     1201 caggttaata tgggtttccg gtttcacttc cggcttgact tccggctttg cttcttggtt
     1261 gacttccgaa tcttgcaggc taccgtggtg atggtgatgg tgatgatggt gatgaatctt
     1321 cgccatcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     1381 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatcgatc
     1441 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     1501 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1561 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1621 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     1681 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     1741 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1801 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1861 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1921 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     1981 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     2041 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     2101 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     2161 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     2221 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     2281 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     2341 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     2401 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     2461 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     2521 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     2581 cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg ggatacgacg ataccgaaga
     2641 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     2701 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     2761 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2821 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2881 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     2941 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     3001 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     3061 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     3121 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     3181 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     3241 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     3301 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     3361 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     3421 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     3481 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3541 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3601 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     3661 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     3721 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     3781 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     3841 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3901 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3961 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     4021 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4081 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     4141 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4201 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4261 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4321 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4381 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4441 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4501 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4561 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4621 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4681 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4741 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4801 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4861 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4921 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4981 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     5041 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     5101 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     5161 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     5221 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     5281 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     5341 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     5401 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     5461 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     5521 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5581 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5641 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5701 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5761 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5821 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     5881 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5941 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6001 ccaat
//
