LOCUS       sequence-217483-        6748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2010 Nov 9. 107(45):19338-43."
COMMENT     Sequence Label: EGFP-Rab5A Q79L
FEATURES             Location/Qualifiers
     source          1..6748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(897..899,900..902,903..1613)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(897..899,900..902,903..1613)
                     /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(942..963)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1203..1222)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1550..1571
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2303..2321)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2304..2321)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2341..2358)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2347..2571
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2617..3045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2704..2723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2914..2935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3054..3074)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3059..3388
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3239..3374
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3301..3320
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3455..4249
                     /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(3509..3528)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4119..4138
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4423..4544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4460..4479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4514..4533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4593..4609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4593..4609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4606..4628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4617..4633
                     /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)."
     promoter        complement(join(4641..4647,4648..4665,4666..4671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4686..4707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4824..4841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4995..5583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5075..5094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5754..6545,6546..6614))
                     /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     6377..6396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6615..6719)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttatgg
      901 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      961 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1021 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1081 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1141 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1201 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1261 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1321 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1381 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1441 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1501 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1561 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggatcca
     1621 tggctagtcg aggcgcaaca agacccaacg ggccaaatac tggaaataaa atatgccagt
     1681 tcaaactagt acttctggga gagtccgctg ttggcaaatc aagcctagtg cttcgttttg
     1741 tgaaaggcca atttcatgaa tttcaagaga gtaccattgg ggctgctttt ctaacccaaa
     1801 ctgtatgtct tgatgacact acagtaaagt ttgaaatatg ggatacagct ggtctagaac
     1861 gataccatag cctagcacca atgtactaca gaggagcaca agcagccata gttgtatatg
     1921 atatcacaaa tgaggagtcc tttgcaagag caaaaaattg ggttaaagaa cttcagaggc
     1981 aagcaagtcc taacattgta atagctttat cgggaaacaa ggccgaccta gcaaataaaa
     2041 gagcagtaga tttccaggaa gcacagtcct atgcagatga caatagttta ttattcatgg
     2101 agacatccgc taaaacatca atgaatgtaa atgaaatatt catggcaata gctaaaaaat
     2161 tgccaaagaa tgaaccacaa aatccaggag caaattctgc cagaggaaga ggagtagacc
     2221 ttaccgaacc cacacaacca accaggaatc agtgttgtag taactgaggc ggccgctcga
     2281 gcatgcatct agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag
     2341 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2401 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2461 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2521 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2581 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2641 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2701 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2761 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2821 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2881 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2941 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3001 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3061 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3121 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3181 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3241 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3301 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3361 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     3421 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3481 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3541 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3601 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3661 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3721 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3781 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3841 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3901 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3961 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4021 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4081 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4141 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4201 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4261 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4321 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4381 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4441 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4501 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4561 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4621 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4681 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5161 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5401 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5641 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5701 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5761 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5821 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5881 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5941 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6001 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6061 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6721 acatttcccc gaaaagtgcc acctgacg
//
