LOCUS       sequence-474399-        6907 bp    DNA     circular SYN 24-JUN-2025
DEFINITION  Negative 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 Jun 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-neg-2H11
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1017..1208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(1790..1817)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1909..1995)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2148..2165
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2433..3146)
                     /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(3507..3593)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3623..3641)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4610..5707
                     /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      5564..5573
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5681..5704
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     promoter        5772..5876
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5877..5945,5946..6737)
                     /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(6095..6114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     1081 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     1141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     1201 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     1261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     1321 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     1381 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     1441 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     1501 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     1561 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     1621 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
     1681 gacccaacgc tgcccgactg ctttcctgat gcaaaaacga ggctagttta ccgtatctgt
     1741 ggggggatgg cttgtagata tgacgacagg aagagtttgt agaaacgcaa aaaggccatc
     1801 cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg
     1861 ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact
     1921 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     1981 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac
     2041 catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc
     2101 tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta
     2161 tcaggctgaa aatcttctct catccgccaa aacagccgag atgcgccgcg tgcggctgct
     2221 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     2281 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgcct
     2341 cgacgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt
     2401 ttaagggcac caataactgc cttaaaaact catttgtaca gttcatccat accatgcgtg
     2461 atgcccgctg cggttacgaa ctccagcaga accatgtgat cgcgtttctc gttcggatct
     2521 ttagacagaa cgctttgcgt gctcagatag tgattgtctg gcagcagaac aggaccatca
     2581 ccgattggag tgttttgctg gtagtgatca gccagctgca cgctgccatc ctccacgttg
     2641 tggcgaattt taaaattcgc tttaatgcca tttttttgtt tatcggcggt gatgtaaaca
     2701 ttgtggctgt taaaattgta ttccagctta tggcccagga tattgccgtc ttctttaaag
     2761 tcaatgcctt tcagctcaat gcggtttacc agggtatcgc cttcaaattt cacttccgca
     2821 cgcgttttgt acgtgccgtc atccttaaag gaaatcgtgc gttcctgcac atagccttcc
     2881 ggcatggcgg acttgaagaa gtcatgctgc ttcatgtggt ccggataacg agcaaagcac
     2941 tgaacaccat aagtcagcgt cgttaccaga gtcggccaag gtaccggcag tttaccagta
     3001 gtacagatga acttcagcgt cagtttacca ttagttgcgt caccttcacc ctcgccacgc
     3061 acggaaaact tatgaccgtt gacatcacca tccagttcca ccagaatagg gacgacacca
     3121 gtgaacagct cttcgccttt gcccatttta gcttccttag ctcctgaaaa tctcgtcgaa
     3181 gctcggcgga tttgtcctac tcaagcttgc tccctttgtt taacagaaaa ataattgatc
     3241 aaaacaatta aataagtaaa taaatttgta ttatttgaaa tcaaaaatca aaaatcaaaa
     3301 atcaaaaatc aaaaatcaaa aatcaaaata tactatttag tcctttttta tcattttctg
     3361 tttttatcgg taaaccccgt ttcacttcta agcttggctg caggtcgacg gatcgatccc
     3421 cgggaattcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
     3481 gcaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
     3541 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg
     3601 gggaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3661 ataataatgg tttcttaggg gcgcgtttct ttattagtgg ttgcagtctc gctcataatc
     3721 gctccgttta cttctgttcc aaacaattga tccattgaga ctcaatggaa ttaccttgat
     3781 gtgcaagtga gatatggaca aaaaatgtaa attcaaggtc aaaactcata aaaacactgt
     3841 tttttgatcg agattggatt attctaagtc tgcattttta tcaaagaaga taaaaaaacc
     3901 agtaaagtct gagtgttggg acagggagat actgggacat tagatgttcg gattaggaca
     3961 caactcaaaa gagatttcga tgagtcatat tggtactaaa ttcattcttg ctgaaaaatt
     4021 taccttcgat cccctaagca atactctgat tgacaaagaa gatagtgaag agatcattcg
     4081 attaggcagc aacgaaagcc gaattctttg gctgctggcc caacgtccaa acgaggtgat
     4141 ttctcgcaat gatttgcatg actttgtttg gcgagagcaa ggttttgaag tcgatgattc
     4201 cagcttaacc caagccattt cgactctgcg caaaatgctc aaagattcga caaagtcccc
     4261 acaatacgtc aaaacggttc cgaagcgcgg ttaccaattg atcgcccgag tggaaacggt
     4321 tgaagaagag atggctcgcg aaaacgaagc tgctcatgac atctctcagc cagaatctgt
     4381 caatgaatac gcagaatcaa gcagtgtgcc ttcatcagcc actgtagtga acacaccgca
     4441 gccagccaat gtcgtggcga ataaatcggc tccaaacttg gggaatcgag ctagcggcat
     4501 ttattttgtg ctgggcgtgt gctttggcct gctgctgacc ctgtgcctgc tggtgatttt
     4561 gatcctgatc aacccaagcc aatccagctt taaaccccta acggatatca aaatcgaaga
     4621 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
     4681 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
     4741 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
     4801 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
     4861 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
     4921 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
     4981 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
     5041 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
     5101 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
     5161 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
     5221 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
     5281 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
     5341 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     5401 aggtattaac gccgccagtc cgaacaaaga gctggcgaaa gagttcctcg aaaactatct
     5461 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     5521 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca tggaaaacgc
     5581 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     5641 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     5701 gcagactaat tcgagctcgg tacccggccg gggatcttaa tctagcaggt ggcacttttc
     5761 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5821 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5881 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5941 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6001 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6061 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     6121 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6181 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6241 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6301 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6361 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6421 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6481 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6541 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6601 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6661 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6721 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6781 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6841 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6901 gatcttc
//
