LOCUS       sequence-464184-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of protein virC2 in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5960)
  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_2RH3
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..36
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(160..207)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     203..221
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(274..291)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(979..1008)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1031..1053
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1068..1092
                     /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(1092..1111)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1093..1111)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1199..1218)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1424..1501
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1502..2584
                     /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(1521..1540)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3393..3584
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3578..3600)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3686..3825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3741..3760
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3840..3857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4011..4599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4091..4110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4710..4729)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4721..5536
                     /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             4721..5536
                     /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(4794..4813)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5628..5960,1..123))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5765..5786)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
       61 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
      121 cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg
      181 tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc
      241 ttcctttcgg gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagtca
      301 ttacgaaccg ctattggtcg ctttttcgcg tgcaaagaag caggccagcg ccgcagtcgc
      361 cagtttatgg ccgaaggctc tagccgtttc caggcccagc gggtcgaagt ggttgcgagc
      421 tgcctcgata aggctcaccg gaaacatacg gctggtctgg acgatgatcg acttctcgaa
      481 cgcggtgtgc ggaatcgggt aggatttcgg tgccgcacga aaactaccat cagccaacat
      541 gttctcaaaa tcacccagcg cacgacgcag aatcatttgc aaggacttgc tcgggctata
      601 ctgtaaaatc aggttgtcgt agatcttaga aacttctggc gcgggtggcc tagctgatag
      661 aaatacttgt attccagagc cgccaatctg ttcacggtgg gcttcaatga tgtcgttgtc
      721 ttccatgtcc aggtcttccg gcgcttgatc tgcctggatg cgaataccgt catacaggaa
      781 acgcaggcta tccatttcct tgccctgacg tttggcaaac gcttccatca ggcgacgcag
      841 cggcgtggtc tttttgatct taaagaaaat ttcagagctg ccgtcactaa ctttcaggtt
      901 aatatgggtt tccggtttca cttccggctt gacttccggc tttgcttctt ggttgacttc
      961 cgaatcttgc aggctaccgt ggtgatggtg atggtgatga tggtgatgaa tcttcgccat
     1021 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1081 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     1141 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1201 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1261 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     1321 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     1381 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     1441 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1501 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1561 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1621 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1681 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1741 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1801 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1861 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1921 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1981 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     2041 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2101 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2161 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2221 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2281 cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg aagacagctc
     2341 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2401 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2461 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2521 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2581 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     2641 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     2701 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     2761 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     2821 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     2881 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     2941 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     3001 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     3061 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     3121 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3181 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3241 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     3301 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     3361 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     3421 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     3481 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3541 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3601 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3661 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3721 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg
     3781 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     3841 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3901 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3961 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4021 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4081 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4141 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4201 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4261 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4321 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4381 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4441 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4501 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4561 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4621 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat
     4681 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4741 aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     4801 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     4861 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     4921 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     4981 tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt
     5041 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     5101 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     5161 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     5221 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     5281 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     5341 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     5401 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     5461 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     5521 cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa
     5581 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5641 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5701 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5761 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5821 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5881 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5941 ttgacgggga aagccggcga
//
