LOCUS       sequence-464194-        5951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of replication protein A 70 kDa 
            DNA-binding subunit in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5951)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5951)
  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_4IPC
FEATURES             Location/Qualifiers
     source          1..5951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(93..114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     305..324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(448..495)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     491..509
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(562..579)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1258..1287)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1310..1332
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1347..1371
                     /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(1371..1390)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1372..1390)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1478..1497)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1703..1780
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1781..2863
                     /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(1800..1819)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3672..3863
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3857..3879)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3965..4104
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4020..4039
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4119..4136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4290..4878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4370..4389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4989..5008)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5000..5815
                     /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             5000..5815
                     /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(5073..5092)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5907..5951,1..411))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
       61 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
      121 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
      181 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
      241 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
      301 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
      361 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
      421 caatccggat atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc
      481 aaggggttat gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc
      541 tttgttagca gccggatctc agtggtggtg gtggtggtgc tcgagtcatt acgaaccgta
      601 cggcaccggg ttgccgattt ttacacccac agcctccgcc gacttcagga cttctaattc
      661 catcaggata acaacacgac ggccgtcctt caacgtattc acgatgaagc ggtgaatctg
      721 gcacacacaa ttgctggaca gttgctcctc ttcaaccagc gggttcaact gggtggcgag
      781 cataaaagag ctcagggtat tcagaccgtc gctcatcagc aagcgataac gcggtgggct
      841 gttaccagtg gtgatcggac gaatgttgat aacctgcaga atcggtttga tgttggtgtc
      901 gcctttttgc ataattgccg ctatagcacc ccttgatagt tgtcctacca ttccagagcc
      961 gccaatctgt tcacggtggg cttcaatgat gtcgttgtct tccatgtcca ggtcttccgg
     1021 cgcttgatct gcctggatgc gaataccgtc atacaggaaa cgcaggctat ccatttcctt
     1081 gccctgacgt ttggcaaacg cttccatcag gcgacgcagc ggcgtggtct ttttgatctt
     1141 aaagaaaatt tcagagctgc cgtcactaac tttcaggtta atatgggttt ccggtttcac
     1201 ttccggcttg acttccggct ttgcttcttg gttgacttcc gaatcttgca ggctaccgtg
     1261 gtgatggtga tggtgatgat ggtgatgaat cttcgccatc atatgtatat ctccttctta
     1321 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     1381 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
     1441 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     1501 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     1561 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     1621 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     1681 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     1741 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     1801 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     1861 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     1921 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1981 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     2041 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     2101 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     2161 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     2221 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     2281 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     2341 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     2401 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     2461 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     2521 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     2581 catctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     2641 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     2701 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     2761 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2821 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2881 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     2941 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     3001 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     3061 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     3121 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     3181 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     3241 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     3301 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3361 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     3421 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3481 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3541 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3601 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     3661 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     3721 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3781 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     3841 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3901 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3961 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     4021 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     4081 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4921 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     4981 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     5041 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5101 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     5161 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5221 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     5281 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     5341 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     5401 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     5461 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     5521 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     5581 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5641 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5701 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5761 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     5821 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5881 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     5941 gttaaatttt t
//
