LOCUS       sequence-464138-        6299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of alpha-actinin-1 in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6299)
  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_2EYI
FEATURES             Location/Qualifiers
     source          1..6299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             586..603
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(656..674)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      670..717
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      754..1209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(841..860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1051..1072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1301..2116)
                     /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(1301..2116)
                     /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     2024..2043
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2108..2127
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2238..2826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2727..2746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2980..2997
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3012..3151
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3077..3096)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3237..3259
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3253..3444)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4253..5335)
                     /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     5297..5316
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5336..5413)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5619..5638
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5726..5745
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5726..5744
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5745..5769
                     /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             5784..5806
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5829..5858
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 gttattagcg gtgaacgcct ggcaaagccg gaacgtggta agatgcgtgt tcataaaatc
       61 tccaacgtga ataaagcgct ggactttatc gcgagcaaag gcgttaaatt ggttagcatt
      121 ggtgctgagg agatcgtaga tggcaacgtg aaaatgaccc tgggcatgat ctggaccatt
      181 atcctgcgtt ttgcgatcca agatatctcc gtggaagaaa cctctgcaaa agaaggtttg
      241 ctattatggt gccagcgtaa gaccgcccct tataaaaacg tcaacattca aaacttccac
      301 atttcttgga aagacggcct gggtttctgc gcgcttatcc accgtcaccg cccagaactg
      361 atcgattatg gcaagctgcg taaggacgac ccgctgacca atctgaatac cgcgtttgat
      421 gttgcggaga agtacctgga catcccgaag atgcttgacg ctgaggacat tgtgggtact
      481 gcgcgtccgg atgaaaaggc cattatgacg tacgtgtcga gcttttacca tgcattcagc
      541 ggcgcacagg agttcctgga gccgggtggt tcgtaatgac tcgagcacca ccaccaccac
      601 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      661 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      721 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      961 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1021 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1081 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1201 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1261 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1321 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1381 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1441 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1501 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1561 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1621 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1681 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1741 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1801 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1861 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1921 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1981 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2041 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2101 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2161 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2221 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2281 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2341 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2401 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2461 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2521 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2581 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2641 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2701 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2761 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2821 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2881 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2941 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3001 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3061 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3121 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3181 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3241 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3301 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3361 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3421 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3481 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3541 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3601 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3661 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3721 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     3781 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     3841 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     3901 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     3961 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4021 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4081 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4141 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4201 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4261 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4321 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4381 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4441 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4501 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     4561 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4621 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4681 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4741 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4801 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4861 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4921 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4981 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5041 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5101 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5161 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5221 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5281 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5341 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5401 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5461 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5521 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5581 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5641 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5701 gagatcgatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     5761 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgatgg
     5821 cgaagattca tcaccatcat caccatcacc atcaccacgg tagcctgcaa gattcggaag
     5881 tcaaccaaga agcaaagccg gaagtcaagc cggaagtgaa accggaaacc catattaacc
     5941 tgaaagttag tgacggcagc tctgaaattt tctttaagat caaaaagacc acgccgctgc
     6001 gtcgcctgat ggaagcgttt gccaaacgtc agggcaagga aatggatagc ctgcgtttcc
     6061 tgtatgacgg tattcgcatc caggcagatc aagcgccgga agacctggac atggaagaca
     6121 acgacatcat tgaagcccac cgtgaacaga ttggcggctc tggagctgga cacatggaaa
     6181 aacagcaaag gaagacgttt accgcttggt gtaatagcca tctgagaaag gcgggtacac
     6241 agattgagaa catcgaagag gatttccgtg acggtctgaa actgatgttg ctcttggag
//
