LOCUS       sequence-464154-        5927 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Heterologous protein expression of disks large homolog 4 (PSD-95) 
            (SAP90) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5927)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAIP4_5HFD
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     642..661
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     726..745
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      856..1444
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1345..1364
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1598..1615
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1630..1769
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1695..1714)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1855..1877
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1871..2062)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2871..3953)
                     /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     3915..3934
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3954..4031)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4237..4256
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4344..4363
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4344..4362
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4363..4387
                     /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             4402..4424
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4447..4476
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5131..5148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5201..5219)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5215..5262
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5299..5754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5386..5405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5596..5617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5846..5927,1..734))
                     /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(5846..5927,1..734))
                     /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 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
       61 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
      121 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
      181 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
      241 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
      301 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
      361 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
      421 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
      481 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
      541 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
      601 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
      661 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
      721 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
      781 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
      841 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
      901 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      961 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1021 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1081 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1141 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1201 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1261 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1321 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1381 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1441 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1501 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1561 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1621 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     1681 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     1741 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     1801 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     1861 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     1921 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     1981 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     2041 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     2101 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     2161 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     2221 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     2281 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     2341 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     2401 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     2461 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     2521 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     2581 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     2641 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     2701 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     2761 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     2821 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     2881 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2941 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     3001 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     3061 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     3121 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     3181 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     3241 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     3301 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     3361 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     3421 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     3481 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     3541 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     3601 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     3661 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     3721 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     3781 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     3841 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     3901 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     3961 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     4021 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     4081 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     4141 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     4201 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     4261 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     4321 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     4381 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgatggcg
     4441 aagattcatc accatcatca ccatcaccat caccacggta gcctgcaaga ttcggaagtc
     4501 aaccaagaag caaagccgga agtcaagccg gaagtgaaac cggaaaccca tattaacctg
     4561 aaagttagtg acggcagctc tgaaattttc tttaagatca aaaagaccac gccgctgcgt
     4621 cgcctgatgg aagcgtttgc caaacgtcag ggcaaggaaa tggatagcct gcgtttcctg
     4681 tatgacggta ttcgcatcca ggcagatcaa gcgccggaag acctggacat ggaagacaac
     4741 gacatcattg aagcccaccg tgaacagatt ggcggctctg gagatattcc cagagaacca
     4801 cgaaggatag taatccaccg cggttcgacc ggtctgggtt ttaacattgt cggaaccgaa
     4861 gacggcgagg gcattttcat cagctttatc ctggcaggcg gtccggcgga tttgtctggc
     4921 gagctgcgta agggtgatca gatcttgtct gttaatggtg ttgacctccg caacgcgagc
     4981 gcggaacaag cggcaattgc gctgaaaaac gctggtcaga ccgtgacgat cattgcgcaa
     5041 tataaaccgg aagagtacag ccgtttcgag gctaatagcc gtgttgacag ctccggccgt
     5101 attgtgactg atggttcgta atgactcgag caccaccacc accaccactg agatccggct
     5161 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     5221 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     5281 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5341 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5401 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5461 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5521 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5581 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5641 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5701 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5761 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5821 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     5881 atttattcat atcaggatta tcaataccat atttttgaaa aagccgt
//
