LOCUS       sequence-464202-        6329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of variant of super folded GFP in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6329)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6329)
  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_9C74
FEATURES             Location/Qualifiers
     source          1..6329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(302..1384)
                     /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     1346..1365
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1385..1462)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1668..1687
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1775..1794
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1775..1793
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1794..1818
                     /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             1833..1855
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1878..1907
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2232..2945
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2964..2981
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3034..3052)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3048..3095
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3132..3587
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3219..3238)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3429..3450
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3679..4494)
                     /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(3679..4494)
                     /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     4402..4421
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4486..4505
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4616..5204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5105..5124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5358..5375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5390..5529
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5455..5474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5615..5637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5631..5822)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
       61 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
      121 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
      181 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
      241 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
      301 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      361 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
      421 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
      481 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
      541 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
      601 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
      661 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
      721 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
      781 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
      841 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
      901 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
      961 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1021 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1081 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1141 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1201 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     1261 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     1321 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     1381 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     1441 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     1501 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     1561 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     1621 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     1681 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     1741 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1801 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1861 atatgatggc gaagattcat caccatcatc accatcacca tcaccacggt agcctgcaag
     1921 attcggaagt caaccaagaa gcaaagccgg aagtcaagcc ggaagtgaaa ccggaaaccc
     1981 atattaacct gaaagttagt gacggcagct ctgaaatttt ctttaagatc aaaaagacca
     2041 cgccgctgcg tcgcctgatg gaagcgtttg ccaaacgtca gggcaaggaa atggatagcc
     2101 tgcgtttcct gtatgacggt attcgcatcc aggcagatca agcgccggaa gacctggaca
     2161 tggaagacaa cgacatcatt gaagcccacc gtgaacagat tggcggctct ggaagagaac
     2221 aaataggagg gatgaggaaa ggtgaggaac tgtttaccgg cgtggtcccg attctggttg
     2281 aactggatgg cgacgttaac ggtcataagt tttctgttcg tggtgaaggc gagggtgacg
     2341 ctaccaatgg caagctcacg ctgaagttca tctgcaccac cggtaagttg ccagttccgt
     2401 ggccgaccct tgtaactacg ctgacctatg gtgttcaatg ttttgcgcgt tatccggatc
     2461 acatgaaaca acatgatttc ttcaagagcg caatgccgga aggttacgtg caagagcgca
     2521 ccattagctt taaggacgat ggcacgtaca agacccgcgc tgaagtgaaa ttcgagggtg
     2581 ataccttagt caatagaatt gaactgaaag gtatcgactt caaagaggat ggaaacattc
     2641 tgggtcacaa attggagtac aacttcaaca gccataatgt gtatattacc gcggacaaac
     2701 agaaaaacgg tatcaaagcg aacttcaaga tccgtcataa cgtcgaggac ggcagcgttc
     2761 agctggcgga ccactaccag cagaataccc cgatcggcga cggccctgtg ctgctgccgg
     2821 acaaccacta tctgtccacc cagtccgtgt tgtcgaaaga cccgaatgaa aaacgtgatc
     2881 acatggtttt gctggaattt gtgactgccg caggcatcac gcacggtatg gatgagctat
     2941 acaagggttc gtaatgactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     3001 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     3061 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3121 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3181 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3241 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3301 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3361 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3421 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3481 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3541 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3601 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3661 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     3721 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     3781 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     3841 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     3901 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     3961 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4021 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     4081 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4141 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4201 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4261 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4321 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4381 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4441 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     4501 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     4561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5401 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5461 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5521 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5581 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5641 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5701 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5761 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5821 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5881 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5941 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     6001 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     6061 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     6121 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     6181 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     6241 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     6301 ccggcgataa tggcctgctt ctcgccgaa
//
