LOCUS       sequence-480239-        6907 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  Positive control for liquid MalE complementation assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   Anderson SM, Choi J, Cushman EM, Leander M, Raman S, Senes A
  TITLE     High-throughput discovery of transmembrane helix dimers from human 
            single-pass membrane proteins with TOXGREEN sort-seq
  JOURNAL   bioRxiv 2025.04.22.650048
REFERENCE   2  (bases 1 to 6907)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.04.22.650048"
COMMENT     Sequence Label: pccGFP-MalE-pos-2H01
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     343..362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     596..613
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    628..768
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(694..713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     854..876
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(870..1061)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(1643..1670)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1762..1848)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2001..2018
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2286..2999)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MGKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      complement(3360..3446)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3476..3494)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4463..5560
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      5417..5426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5534..5557
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     promoter        5625..5729
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5730..5798,5799..6590)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5948..5967)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6761..6907,1..442)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
       61 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      121 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      181 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      241 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      301 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      361 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      421 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      481 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      541 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      601 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
      661 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      721 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
      781 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      841 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
      901 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
      961 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     1021 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     1081 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     1141 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     1201 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     1261 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     1321 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     1381 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     1441 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     1501 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga ctgctttcct
     1561 gatgcaaaaa cgaggctagt ttaccgtatc tgtgggggga tggcttgtag atatgacgac
     1621 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg
     1681 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac
     1741 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     1801 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt
     1861 tccctactct cgcatgggga gaccccacac taccatcggc gctacggcgt ttcacttctg
     1921 agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa ttctgtttta
     1981 tcagaccgct tctgcgttct gatttaatct gtatcaggct gaaaatcttc tctcatccgc
     2041 caaaacagcc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt
     2101 ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct
     2161 tggagtggtg aatccgttag cgaggtgccg cctcgacgaa tttctgccat tcatccgctt
     2221 attatcactt attcaggcgt agcaaccagg cgtttaaggg caccaataac tgccttaaaa
     2281 actcatttgt acagttcatc cataccatgc gtgatgcccg ctgcggttac gaactccagc
     2341 agaaccatgt gatcgcgttt ctcgttcgga tctttagaca gaacgctttg cgtgctcaga
     2401 tagtgattgt ctggcagcag aacaggacca tcaccgattg gagtgttttg ctggtagtga
     2461 tcagccagct gcacgctgcc atcctccacg ttgtggcgaa ttttaaaatt cgctttaatg
     2521 ccattttttt gtttatcggc ggtgatgtaa acattgtggc tgttaaaatt gtattccagc
     2581 ttatggccca ggatattgcc gtcttcttta aagtcaatgc ctttcagctc aatgcggttt
     2641 accagggtat cgccttcaaa tttcacttcc gcacgcgttt tgtacgtgcc gtcatcctta
     2701 aaggaaatcg tgcgttcctg cacatagcct tccggcatgg cggacttgaa gaagtcatgc
     2761 tgcttcatgt ggtccggata acgagcaaag cactgaacac cataagtcag cgtcgttacc
     2821 agagtcggcc aaggtaccgg cagtttacca gtagtacaga tgaacttcag cgtcagttta
     2881 ccattagttg cgtcaccttc accctcgcca cgcacggaaa acttatgacc gttgacatca
     2941 ccatccagtt ccaccagaat agggacgaca ccagtgaaca gctcttcgcc tttgcccatt
     3001 ttagcttcct tagctcctga aaatctcgtc gaagctcggc ggatttgtcc tactcaagct
     3061 tgctcccttt gtttaacaga aaaataattg atcaaaacaa ttaaataagt aaataaattt
     3121 gtattatttg aaatcaaaaa tcaaaaatca aaaatcaaaa atcaaaaatc aaaaatcaaa
     3181 atatactatt tagtcctttt ttatcatttt ctgtttttat cggtaaaccc cgtttcactt
     3241 ctaagcttgg ctgcaggtcg acggatcgat ccccgggaat tcctggcagt ttatggcggg
     3301 cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga
     3361 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     3421 ttcgactgag cctttcgttt tatttgatgc ctggggaatt cttgaagacg aaagggcctc
     3481 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta ggggcgcgtt
     3541 tctttattag tggttgcagt ctcgctcata atcgctccgt ttacttctgt tccaaacaat
     3601 tgatccattg agactcaatg gaattacctt gatgtgcaag tgagatatgg acaaaaaatg
     3661 taaattcaag gtcaaaactc ataaaaacac tgttttttga tcgagattgg attattctaa
     3721 gtctgcattt ttatcaaaga agataaaaaa accagtaaag tctgagtgtt gggacaggga
     3781 gatactggga cattagatgt tcggattagg acacaactca aaagagattt cgatgagtca
     3841 tattggtact aaattcattc ttgctgaaaa atttaccttc gatcccctaa gcaatactct
     3901 gattgacaaa gaagatagtg aagagatcat tcgattaggc agcaacgaaa gccgaattct
     3961 ttggctgctg gcccaacgtc caaacgaggt gatttctcgc aatgatttgc atgactttgt
     4021 ttggcgagag caaggttttg aagtcgatga ttccagctta acccaagcca tttcgactct
     4081 gcgcaaaatg ctcaaagatt cgacaaagtc cccacaatac gtcaaaacgg ttccgaagcg
     4141 cggttaccaa ttgatcgccc gagtggaaac ggttgaagaa gagatggctc gcgaaaacga
     4201 agctgctcat gacatctctc agccagaatc tgtcaatgaa tacgcagaat caagcagtgt
     4261 gccttcatca gccactgtag tgaacacacc gcagccagcc aatgtcgtgg cgaataaatc
     4321 ggctccaaac ttggggaatc gagctagctt tcatatgatt gcggtgggcc tgagcagcag
     4381 cattctgggc tgcctgatta ccctgctggt gttgatcctg atcaacccaa gccaatccag
     4441 ctttaaaccc ctaacggata tcaaaatcga agaaggtaaa ctggtaatct ggattaacgg
     4501 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
     4561 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
     4621 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
     4681 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
     4741 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
     4801 atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc
     4861 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
     4921 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa
     4981 cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac
     5041 cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc
     5101 agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc
     5161 caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca
     5221 accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa
     5281 agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt
     5341 taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa
     5401 agatccacgt attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat
     5461 cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg
     5521 tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cggtacccgg
     5581 ccggggatct taatctagca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5641 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5701 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5761 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5821 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5881 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5941 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     6001 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6061 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6121 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6181 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6241 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     6301 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6361 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6421 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6481 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6541 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6601 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6661 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6721 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6781 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6841 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6901 atactgt
//
