LOCUS       sequence-464166-        6035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of multivesicular body subunit 12B 
            (ESCRT-I complex subunit MVB12B) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6035)
  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_3TOW
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     696..715
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     780..799
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1652..1669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1684..1823
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1749..1768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1909..1931
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1925..2116)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2925..4007)
                     /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     3969..3988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4008..4085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4291..4310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4398..4417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4398..4416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4417..4441
                     /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             4456..4478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4501..4530
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5293..5310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5363..5381)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5377..5424
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5461..5916
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5548..5567)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5758..5779
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6008..6035,1..788))
                     /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(join(6008..6035,1..788))
                     /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"
ORIGIN
        1 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
       61 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
      121 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
      181 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
      241 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
      301 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
      361 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
      421 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
      481 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
      541 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
      601 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
      661 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
      721 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
      781 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     1621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     1681 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     1741 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     1801 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     1861 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1921 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     1981 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2041 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2101 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     2161 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     2221 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     2281 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     2341 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     2401 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     2461 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     2521 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     2581 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     2641 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     2701 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     2761 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     2821 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     2881 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     2941 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3001 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3061 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3181 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     3241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     3421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     3481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     3541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     3601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     3661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     3721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     3781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     3841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     3901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     3961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     4141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     4201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     4261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     4321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     4381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     4441 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgat ggcgaagatt
     4501 catcaccatc atcaccatca ccatcaccac ggtagcctgc aagattcgga agtcaaccaa
     4561 gaagcaaagc cggaagtcaa gccggaagtg aaaccggaaa cccatattaa cctgaaagtt
     4621 agtgacggca gctctgaaat tttctttaag atcaaaaaga ccacgccgct gcgtcgcctg
     4681 atggaagcgt ttgccaaacg tcagggcaag gaaatggata gcctgcgttt cctgtatgac
     4741 ggtattcgca tccaggcaga tcaagcgccg gaagacctgg acatggaaga caacgacatc
     4801 attgaagccc accgtgaaca gattggcggc tctggaatgg atcccataac aggggtcgga
     4861 gtagttgcga gccgtaaccg tgcaccgacg ggttatgatg ttgttgcgca gaccgctgat
     4921 ggcgttgacg ctgatctgtg gaaagacggc ttgttcaagt cgaaggtgac ccgctacctg
     4981 tgttttaccc gctccttcag caaagagaac tcccacctgg gtaacgtgct ggtcgacatg
     5041 aagctgattg acatcaagga cacgctgccg gttggtttta tcccgattca agagactgtg
     5101 gacacccaag aggtggcctt tcgtaaaaaa cgtttatgca ttaagttcat cccgagagat
     5161 agcaccgaag cggcaatttg cgatatccgc atcatgggcc gtaccaaaca ggcgccaccg
     5221 cagtacacct tcatcggcga attgaattct atgggtattt ggtatcgtat gggtggttcg
     5281 taatgactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     5341 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     5401 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     5461 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5521 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5581 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5641 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5701 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5761 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5821 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5881 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     6001 ttaattctta gaaaaactca tcgagcatca aatga
//
