LOCUS       sequence-464140-        6113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of uncharacterized protein in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6113)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6113)
  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_4O6G
FEATURES             Location/Qualifiers
     source          1..6113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(84..131)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     127..145
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(198..215)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1056..1085)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1108..1130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1145..1169
                     /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(1169..1188)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1170..1188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1276..1295)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1501..1578
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1579..2661
                     /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(1598..1617)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3470..3661
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3655..3677)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3763..3902
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3818..3837
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3917..3934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4088..4676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4168..4187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4787..4806)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4798..5613
                     /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             4798..5613
                     /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(4871..4890)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5705..6113,1..47))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5842..5863)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6054..6073
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat
       61 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
      121 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
      181 ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gtcattacga accccacagg
      241 gtgaacagcg gtttgccctc ggagtccaga aaggagtctt tgataacatt aacgctgacg
      301 tccaggtagt ggctcagctc aaccaaacga tccgccgcgg taatcgccac gacggtgccg
      361 gaagagtcaa ttaaggtatc acgctggcgg ccagcaaagg tcatcgtacc aatgctatac
      421 tccggcatca gttcctctgg ttggaacggt gcgcggattg ccggaagctc acgttcgcta
      481 cgcaccgagc caccgaagta gccgtacaag tacttttcga tcacgctcat gctggcggcc
      541 gcaaattcat aaccttcacg gctcatgcga tcgcttgaag tgataacata ccaaccagcc
      601 aggcggtcga tgaaataacg cacttcacca cccttgttcc acaggatggt acgaccatcg
      661 ttcgtctcac taccttgaat catgttcatg ccagacaatc taatccaatc ctgcaggtct
      721 gttgatagat caactcctat agttacactt ccagagccgc caatctgttc acggtgggct
      781 tcaatgatgt cgttgtcttc catgtccagg tcttccggcg cttgatctgc ctggatgcga
      841 ataccgtcat acaggaaacg caggctatcc atttccttgc cctgacgttt ggcaaacgct
      901 tccatcaggc gacgcagcgg cgtggtcttt ttgatcttaa agaaaatttc agagctgccg
      961 tcactaactt tcaggttaat atgggtttcc ggtttcactt ccggcttgac ttccggcttt
     1021 gcttcttggt tgacttccga atcttgcagg ctaccgtggt gatggtgatg gtgatgatgg
     1081 tgatgaatct tcgccatcat atgtatatct ccttcttaaa gttaaacaaa attatttcta
     1141 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
     1201 cgagatcgat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1261 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1321 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1381 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1441 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1501 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1561 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1621 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1681 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     1741 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     1801 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     1861 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     1921 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     1981 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     2041 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     2101 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     2161 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     2221 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     2281 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     2341 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt gggatacgac
     2401 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2461 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2521 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2581 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2641 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     2701 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2761 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2821 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     2881 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     2941 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     3001 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     3061 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     3121 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     3181 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     3241 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     3301 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3361 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     3421 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     3481 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     3541 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     3601 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3661 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3721 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     3781 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     3841 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3901 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3961 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4021 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4081 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4141 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4201 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4261 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4321 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4381 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4441 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4501 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4561 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4621 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4681 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4741 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     4801 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     4861 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     4921 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     4981 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     5041 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     5101 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     5161 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     5221 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     5281 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     5341 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     5401 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     5461 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     5521 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     5581 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     5641 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5701 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5761 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5821 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5881 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5941 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     6001 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     6061 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcg
//
