LOCUS       sequence-425978-        6579 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of full-length human RAB7A. C-terminal NanoLuc 
            luciferase tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  AUTHORS   Ackloo S, Li F, Szewczyk M, Seitova A, Loppnau P, Zeng H, Xu J, 
            Ahmad S, Arnautova Y, Baghaie A, Beldar S, Bolotokova A, Centrella 
            P, Chau I, Clark M, Cuozzo J, Dehghani-Tafti S, Disch J, Dong A, 
            Dumas A, Feng J, Ghiabi P, Gibson E, Gilmer J, Goldman B, Green S, 
            Guie M, Guilinger J, Harms N, Herasymenko O, Houliston S, Hutchinson
            A, Kearnes S, Keefe A, Kimani S, Kramer T, Kutera M, Kwak H, Lento 
            C, Li Y, Liu J, Loup J, Machado R, Mulhern C, Perveen S, Righetto G,
            Riley P, Shrestha S, Sigel E, Silva M, Sintchak M, Slakman B, Taylor
            R, Thompson J, Torng W, Underkoffler C, Rechenberg M, Watson I, 
            Wilson D, Wolf E, Yadav M, Yazdi A, Zhang J, Zhang Y, Santhakumar V,
            Edwards A, Barsyte-Lovejoy D, Schapira M, Brown P, Halabelian L, 
            Arrowsmith C
  TITLE     A resource to enable chemical biology and drug discovery of WDR 
            Proteins
  JOURNAL   bioRxiv 2024.03.03.583197
REFERENCE   2  (bases 1 to 6579)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.03.03.583197"
COMMENT     Sequence Label: pNLF1-C_RAB7A:1-207
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1754..2266
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2342..2360)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2397..2518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2408..2427)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2462..2481
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2763..3120
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2763..2954
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2786..2806)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2971..3106
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3151..4188
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4167..4175
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4212..4260
                     /note="synthetic polyadenylation signal"
     promoter        4416..4520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4521..4589,4590..5381)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4739..4758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5539..6127)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5619..5638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6243..6526
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gcgatcgctt ccgaattcgc
     1081 caccatgacc tctaggaaga aagtgttgct gaaggttatc atcctgggag attctggagt
     1141 cgggaagaca tcactcatga accagtatgt gaataagaaa ttcagcaatc agtacaaagc
     1201 cacaatagga gctgactttc tgaccaagga ggtgatggtg gatgacaggc tagtcacaat
     1261 gcagatatgg gacacagcag gacaggaacg gttccagtct ctcggtgtgg ccttctacag
     1321 aggtgcagac tgctgcgttc tggtatttga tgtgactgcc cccaacacat tcaaaaccct
     1381 agatagctgg agagatgagt ttctcatcca ggccagtccc cgagatcctg aaaacttccc
     1441 atttgttgtg ttgggaaaca agattgacct cgaaaacaga caagtggcca caaagcgggc
     1501 acaggcctgg tgctacagca aaaacaacat tccctacttt gagaccagtg ccaaggaggc
     1561 catcaacgtg gagcaggcgt tccagacgat tgcacggaat gcacttaagc aggaaacgga
     1621 ggtggagctg tacaacgaat ttcctgaacc tatcaaactg gacaagaatg accgggccaa
     1681 ggcctcggca gaaagctgca gttgctctag atttgggccc aattcctgca ggcgagctct
     1741 cggctcgagc ggcgtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg
     1801 ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg
     1861 ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga
     1921 catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa
     1981 aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg
     2041 cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga
     2101 aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa
     2161 caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat
     2221 caacggagtg accggctggc ggctgtgcga acgcattctg gcgtaaggcc gcgactctag
     2281 ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2341 accgctgagc aataactagc ataacccctt ggggcggccg cttcgagcag acatgataag
     2401 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2461 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2521 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     2581 aagcaagtaa aacctctaca aatgtggtaa aatcgaattt taacaaaata ttaacgctta
     2641 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     2701 cggatctgcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttaggtacc
     2761 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2821 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2881 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2941 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3001 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3061 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3121 aagctcgatt cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc
     3181 gttgaaaaat ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag
     3241 ggcgaagaga gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg
     3301 aatagctgcg ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct
     3361 gcactaccca tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc
     3421 atcagtagac gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct
     3481 gtgttacagc ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc
     3541 agcggcttcg gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc
     3601 atttgcgcca ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgacaccgtg
     3661 tccgccagcg tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag
     3721 gtgcgccacc tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc
     3781 atcaccgccg taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc
     3841 aacatcttct tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag
     3901 cgccggcatc ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc
     3961 ctggatcagc tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa
     4021 ggccgctgcg atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct
     4081 cgccggagcg cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac
     4141 cgccggccca gtacacgacc gcgcgctaag gaggtaggtc gagtttagac tctagccatc
     4201 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4261 aatcgatagc gataaggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt
     4321 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtaatggttt
     4381 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4441 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4501 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4561 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4621 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4681 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttc aaagttctgc
     4741 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4801 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4861 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4921 acttacttct gacaactatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4981 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5041 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5101 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5161 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5221 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5281 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5341 agatcgctga gataggtgcc tcactgatta agcattggta attcgaaatg accgaccaag
     5401 cgacgcccaa ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5461 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5521 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5581 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5641 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5701 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5761 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5821 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5881 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5941 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     6001 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6061 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6121 atttcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6181 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata
     6241 gtccggaaat acaggaacgc acgctggatg gcccttcgct gggatggtga aaccatgaaa
     6301 aatggcagct tcagtggatt aagtgggggt aatgtggcct gtaccctctg gttgcatagg
     6361 tattcatacg gttaaaattt atcaggcgcg attgcggcag tttttcgggt ggtttgttgc
     6421 catttttacc tgtctgctgc cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta
     6481 agggattatg gtaaatccac ttactgtctg ccctcgtagc catcgagata aaccgcagta
     6541 ctccggccac gatgcgtccg gcgtagagga tcgagatct
//
