LOCUS       sequence-464110-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of GUNA_RUMCH in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6737)
  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_1EDG
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..255
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(249..271)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    357..496
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     412..431
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(511..528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(682..1270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(762..781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1381..1400)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1392..2207
                     /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             1392..2207
                     /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(1465..1484)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2299..2754)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2436..2457)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2648..2667
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2791..2838)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2834..2852
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2905..2922)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4387..4416)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4439..4461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4476..4500
                     /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(4500..4519)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4501..4519)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4607..4626)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4832..4909
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4910..5992
                     /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(4929..4948)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
       61 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
      121 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
      181 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
      241 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
      301 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
      361 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
      421 agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag
      481 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
      541 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      601 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
      901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
      961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1081 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     1141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1261 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1321 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     1381 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     1441 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     1501 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     1561 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     1621 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     1681 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     1741 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     1801 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     1861 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     1921 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     1981 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     2041 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     2101 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     2161 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     2221 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2281 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2341 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2401 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2461 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2521 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2581 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2641 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2701 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     2761 cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc
     2821 cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg
     2881 ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgctcgagtc attacgaacc
     2941 gttgatgagg ccgaaggcgt acttaaccat gccgtcgatg atctccggga atttaaactg
     3001 acagctacga cggtcgaaga aaccaaacag ttcaccagtg ccgctgaagt tattgttatc
     3061 ccacaggatg caaaggatac ctctcgcttt tgcttgcgca acgtaatagc tcatatattc
     3121 cacacgggtt ttcaggttgt tcttgtccac cgcaccgcac tcaccaataa taaccggaat
     3181 accgcgactg gtgtacttgt tatagatgtt atccataaac caggtaactt ccgactgatc
     3241 tttagaatca ttgatattcc acgcgttggt tccgccatcc gccattgcca gaccagcgaa
     3301 attccacggg caatacgcat ggacggacac gatgatcttg ttgttgttgc cggagatgtc
     3361 attcggcatg cggaagtagt cgttggtagc accatctggg ctggccacgt aacccgggca
     3421 catcagatag cgggacgcat tcttaccgcc ggttgcgcga accgtgttca caaagtcttg
     3481 gttcagctgg ttaatacaat taatgctgtc aactacgtcg ctgttggtca actccggcca
     3541 ccactcgttc gcatgaccaa ccaaacgcgg ctcgttcata ccttcaaaga tcaggtgttc
     3601 atcatagttc gcaaaacgag ctgcaatttg tgcccacacc gaggttatgt atttctttga
     3661 ggaagccata tactgagagc ttgggaagta acccttcacc ttgtcaacat cgtggtgggt
     3721 attcagaatc acatacatct tgttgtcaat gcagtaattc acgacctcct ggacacgatt
     3781 catccaaacg tcgctaattt tgtaatcgct gccgctcacg tgcggatgcc agctgaccgg
     3841 aatacgtaca gtgttaaagc ctttttgctt aatcgcgtca atcatctgtt tggtcgtctt
     3901 gatgcctgac cagcttgtct cgtaatctaa ttcattggtg atgttcgtgc cgttgaaggc
     3961 atcaaacgta ttacccaggt tccaacccaa acgcaggcct ttaacgaaat tcatgccgtc
     4021 gttattcggg atgtttttct gcgggatttg tagattgggt attaatgaag catcatacat
     4081 tccagagccg ccaatctgtt cacggtgggc ttcaatgatg tcgttgtctt ccatgtccag
     4141 gtcttccggc gcttgatctg cctggatgcg aataccgtca tacaggaaac gcaggctatc
     4201 catttccttg ccctgacgtt tggcaaacgc ttccatcagg cgacgcagcg gcgtggtctt
     4261 tttgatctta aagaaaattt cagagctgcc gtcactaact ttcaggttaa tatgggtttc
     4321 cggtttcact tccggcttga cttccggctt tgcttcttgg ttgacttccg aatcttgcag
     4381 gctaccgtgg tgatggtgat ggtgatgatg gtgatgaatc ttcgccatca tatgtatatc
     4441 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     4501 cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct ctacgccgga
     4561 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     4621 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     4681 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     4741 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     4801 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     4861 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     4921 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     4981 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     5041 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     5101 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     5161 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     5221 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     5281 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     5341 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     5401 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     5461 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     5521 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     5581 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     5641 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     5701 tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     5761 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     5821 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     5881 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5941 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     6001 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     6061 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     6121 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     6181 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     6241 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     6301 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     6361 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     6421 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     6481 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     6541 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     6601 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     6661 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     6721 cccgtatcgt gagcatc
//
