LOCUS       sequence-464200-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of beta-lactamase in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6371)
  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_3W4Q
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..99)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    185..324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     240..259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(339..356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(510..1098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(590..609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1209..1228)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1220..2035
                     /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             1220..2035
                     /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(1293..1312)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2127..2582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2264..2285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2476..2495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2619..2666)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2662..2680
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2733..2750)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3849..3878)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3901..3923
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3938..3962
                     /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(3962..3981)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3963..3981)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4069..4088)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4294..4371
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4372..5454
                     /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(4391..4410)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             join(6263..6371,1..83)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
       61 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
      121 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
      181 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
      241 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
      301 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
      361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
      421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
      481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
      541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
      601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
      661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
      721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
      781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
      841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
      901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
      961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1141 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     1201 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     1261 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     1321 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     1381 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     1441 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     1501 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     1561 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     1621 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     1681 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     1741 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     1801 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     1861 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     1921 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     1981 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     2041 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2101 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     2161 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2221 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2281 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2341 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2401 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2461 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2521 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2581 gtcccattcg ccaatccgga tatagttcct cctttcagca aaaaacccct caagacccgt
     2641 ttagaggccc caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct
     2701 tcctttcggg ctttgttagc agccggatct cagtggtggt ggtggtggtg ctcgagtcat
     2761 tacgaaccgg ccagagttgc agacgcgata cgagtggcag cggcgatcac atcgtccttc
     2821 gccttcgcgt cagcacgggt ctgggtgtaa tacaccgcca acacgatcgg agcacggctc
     2881 ggaggccata agacaccaac atcgttcgtg gtgccgtagt cgccggtgcc ggttttgtcg
     2941 cccactctcc agccagtcgg aacacccgca cggatgcgct tgtcgcccac tttattacct
     3001 ctcagccatt caatcagttg cgcacgttga gccggtggga gtgcatcacc caaaaccagc
     3061 acgcgcaggt tcgctgccat tgcagccggc gtggtggtat cacgcaggtc acccggcagc
     3121 gcggtattca actcggtttc ccaacgatcc aggcggaagg tgtcgtcgcc aatgctacgt
     3181 gcgtacgccg tgaccgccgc cggaccaccg atacgtttca tcagctcgtt ggcagcggta
     3241 ttatccgaat actgaatggt cgccgcgcac aactccgcaa cggtcatgcc cgtatcaacg
     3301 tgacgttcgg taaccgggga gtagttcacc aggtcggaac gaccatatgt tacacgctgc
     3361 tgcagcagac ccggatgcgc aacgctttga gccaacacag ctgcgcccag cattgcttta
     3421 aaggtgctgc agaacggaaa gcgctcgtcc gcacggtgaa gggcacggcg acccgtcgcg
     3481 gtatcaattg cacaaacacc aaggcgaccg cccgcagccc tttctagtgc tgcaaatgaa
     3541 gctccagagc cgccaatctg ttcacggtgg gcttcaatga tgtcgttgtc ttccatgtcc
     3601 aggtcttccg gcgcttgatc tgcctggatg cgaataccgt catacaggaa acgcaggcta
     3661 tccatttcct tgccctgacg tttggcaaac gcttccatca ggcgacgcag cggcgtggtc
     3721 tttttgatct taaagaaaat ttcagagctg ccgtcactaa ctttcaggtt aatatgggtt
     3781 tccggtttca cttccggctt gacttccggc tttgcttctt ggttgacttc cgaatcttgc
     3841 aggctaccgt ggtgatggtg atggtgatga tggtgatgaa tcttcgccat catatgtata
     3901 tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt
     3961 cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg
     4021 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     4081 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     4141 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     4201 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     4261 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     4321 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     4381 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     4441 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     4501 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     4561 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     4621 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     4681 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     4741 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     4801 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     4861 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     4921 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     4981 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     5041 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     5101 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     5161 gttggtgcgg acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     5221 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     5281 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     5341 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5401 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5461 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     5521 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     5581 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     5641 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     5701 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     5761 cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct
     5821 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     5881 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     5941 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     6001 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     6061 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     6121 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     6181 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     6241 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     6301 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     6361 acaggcagac a
//
