LOCUS       Exported                6748 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    EGFP-Rab5A S34N
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6748)
  AUTHORS   Sun Q, Westphal W, Wong KN, Tan I, Zhong Q
  TITLE     Rubicon controls endosome maturation as a Rab7 effector.
  JOURNAL   Proc Natl Acad Sci U S A. 2010 Nov 9. 107(45):19338-43.
  PUBMED    20974968
REFERENCE   2  (bases 1 to 6748)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6748
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    270..391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(281..300)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     335..354
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(565..1359)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(676..695)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1286..1305
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1426..1755)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1440..1575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1494..1513)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1740..1760
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1769..2197)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1879..1900)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2091..2110
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2243..2467
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2456..2473
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        2493..2511
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2493..2510
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(3201..3917)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(3201..3917)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3243..3264)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3592..3611
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3851..3872
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3930..3949)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3931..3949)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3994..4197)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4023..4043)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4198..4577
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4749..4768
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4843..4947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4948..5808
                     /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(5166..5185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5979..6567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6468..6487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6721..6738
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
      241 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
      301 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      361 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
      421 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
      481 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
      541 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
      601 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
      661 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
      721 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
      781 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
      841 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
      901 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
      961 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     1021 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     1081 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     1141 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     1201 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     1261 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     1321 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     1381 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     1441 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     1501 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     1561 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     1621 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     1681 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     1741 ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca
     1801 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     1861 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     1921 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     1981 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2041 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2101 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2161 acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag
     2221 ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
     2281 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     2341 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     2401 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     2461 ggcacagtcg aggctgatca gcgagctcta gcatttaggt gacactatag aatagggccc
     2521 tctagatgca tgctcgagcg gccgcctcag ttactacaac actgattcct ggttggttgt
     2581 gtgggttcgg taaggtctac tcctcttcct ctggcagaat ttgctcctgg attttgtggt
     2641 tcattctttg gcaatttttt agctattgcc atgaatattt catttacatt cattgatgtt
     2701 ttagcggatg tctccatgaa taataaacta ttgtcatctg cataggactg tgcttcctgg
     2761 aaatctactg ctcttttatt tgctaggtcg gccttgtttc ccgataaagc tattacaatg
     2821 ttaggacttg cttgcctctg aagttcttta acccaatttt ttgctcttgc aaaggactcc
     2881 tcatttgtga tatcatatac aactatggct gcttgtgctc ctctgtagta cattggtgct
     2941 aggctatggt atcgttcttg accagctgta tcccatattt caaactttac tgtagtgtca
     3001 tcaagacata cagtttgggt tagaaaagca gccccaatgg tactctcttg aaattcatga
     3061 aattggcctt tcacaaaacg aagcactagg ctatttttgc caacagcaga ctctcccaga
     3121 agtactagtt tgaactggca tattttattt ccagtatttg gcccgttggg tcttgttgcg
     3181 cctcgactag ccatggatcc cttgtacagc tcgtccatgc cgagagtgat cccggcggcg
     3241 gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg
     3301 gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg
     3361 ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg
     3421 aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg
     3481 tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     3541 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     3601 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac
     3661 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag
     3721 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     3781 ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg
     3841 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     3901 tcgcccttgc tcaccataag cttgggtctc cctatagtga gtcgtattaa tttcgataag
     3961 ccagtaagca gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg
     4021 tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc
     4081 ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa
     4141 tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat
     4201 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
     4261 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     4321 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     4381 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
     4441 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     4501 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     4561 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
     4621 cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta
     4681 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
     4741 taactatgcg gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct
     4801 cccgatccgt cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4861 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4921 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4981 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5041 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5101 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5161 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5221 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5281 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5341 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5401 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5461 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5521 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5581 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5641 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5701 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5761 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5821 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6121 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6601 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6661 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6721 agcgagtcag tgagcgagga agcggaag
//
