LOCUS       sequence-464136-        5900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of synaptojanin-2-binding protein in
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5900)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5900)
  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_2JIK
FEATURES             Location/Qualifiers
     source          1..5900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(118..139)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     330..349
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(473..520)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     516..534
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(587..604)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1232..1261)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1284..1306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1321..1345
                     /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(1345..1364)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1346..1364)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1452..1471)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1677..1754
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1755..2837
                     /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(1774..1793)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3646..3837
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3831..3853)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3939..4078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3994..4013
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4093..4110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4264..4852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4344..4363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4963..4982)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4974..5789
                     /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             4974..5789
                     /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(5047..5066)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5881..5900,1..436))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
       61 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
      121 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
      181 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
      241 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
      301 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
      361 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
      421 ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac
      481 ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag
      541 cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt
      601 ggtgctcgag tcattacgaa ccaatgctgc tttccagacg atgttgaaca cgcagcgaaa
      661 cggcataacc cgcattgcgg aacagatcca cagcgtcttg gtgcagcaag ttcttcaagt
      721 cctggccatt aacagacagg attttatcgc cctcttggag acgaccatcc agtgccgccg
      781 cgccgttctc tttaatgcgg gacacgtaaa taccgctgtc gttggagacg tactgctggt
      841 ccgtgccacc cacgatgtta aaacccaggc cgctcggacc acgggttaag ttgatttcct
      901 cttctgttac tagataatcc attgatccag agccgccaat ctgttcacgg tgggcttcaa
      961 tgatgtcgtt gtcttccatg tccaggtctt ccggcgcttg atctgcctgg atgcgaatac
     1021 cgtcatacag gaaacgcagg ctatccattt ccttgccctg acgtttggca aacgcttcca
     1081 tcaggcgacg cagcggcgtg gtctttttga tcttaaagaa aatttcagag ctgccgtcac
     1141 taactttcag gttaatatgg gtttccggtt tcacttccgg cttgacttcc ggctttgctt
     1201 cttggttgac ttccgaatct tgcaggctac cgtggtgatg gtgatggtga tgatggtgat
     1261 gaatcttcgc catcatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
     1321 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     1381 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     1441 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     1501 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     1561 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     1621 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     1681 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     1741 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     1801 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     1861 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     1921 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     1981 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     2041 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     2101 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     2161 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     2221 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     2281 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     2341 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     2401 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     2461 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     2521 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
     2581 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     2641 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     2701 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     2761 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2821 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     2881 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     2941 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     3001 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     3061 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     3121 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     3181 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     3241 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     3301 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     3361 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     3421 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     3481 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     3541 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     3601 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     3661 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     3721 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     3781 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3841 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3901 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     3961 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     4021 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     4081 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4141 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4201 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4261 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4321 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4381 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4441 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4501 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4561 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4621 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4681 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4741 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4801 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4861 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4921 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4981 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     5041 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     5101 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     5161 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     5221 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     5281 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     5341 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     5401 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     5461 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     5521 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     5581 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     5641 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     5701 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     5761 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     5821 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5881 aaattgtaag cgttaatatt
//
