LOCUS       sequence-364365-        6780 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 6780)
  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 6780)
  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-Rab7A Q67L
FEATURES             Location/Qualifiers
     source          1..6780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..153
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     393..414
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(472..491)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(473..491)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(536..739)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(565..585)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        740..1119
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1291..1310
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1385..1489
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1490..1558,1559..2350)
                     /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(1708..1727)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2521..3109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3010..3029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3263..3280
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3397..3418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3433..3438,3439..3456,3457..3463)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3471..3487
                     /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     3476..3498
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3495..3511
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3495..3511
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3560..3681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3571..3590)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3625..3644
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3855..4649)
                     /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(3966..3985)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4576..4595
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4716..5045)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4730..4865
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4784..4803)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5030..5050
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5059..5487)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5169..5190)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5381..5400
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5533..5757
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5746..5763
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        5783..5801
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5783..5800
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(6460..6486)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6487..6513)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(6523..6780,1..453,454..456,457..459))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6523..6780,1..453,454..456,457..459))
                     /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(6565..6586)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
       61 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
      121 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
      181 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
      241 gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag
      301 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
      361 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
      421 caccaccccg gtgaacagct cctcgccctt gctcaccata agcttgggtc tccctatagt
      481 gagtcgtatt aatttcgata agccagtaag cagtgggttc tctagttagc cagagagctc
      541 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
      601 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
      661 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
      721 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
      781 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
      841 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
      901 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
      961 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     1021 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     1081 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta
     1141 catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca
     1201 agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac
     1261 aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     1321 accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa
     1381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1441 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1501 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1561 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1621 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1681 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1741 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1801 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1861 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1921 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1981 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2041 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2101 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2161 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2221 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2281 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2341 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2401 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2461 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2521 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2581 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2641 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2701 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2761 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2821 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2881 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2941 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3001 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3061 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3121 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3181 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3241 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     3301 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3361 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     3421 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     3481 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     3541 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     3601 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3661 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     3721 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     3781 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     3841 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     3901 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     3961 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     4021 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     4081 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     4141 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     4201 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     4261 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     4321 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     4381 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     4441 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     4501 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     4561 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     4621 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     4681 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     4741 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     4801 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     4861 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     4921 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     4981 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     5041 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5101 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     5161 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     5221 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     5281 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5341 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     5401 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5461 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     5521 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     5581 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     5641 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     5701 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     5761 aggctgatca gcgagctcta gcatttaggt gacactatag aatagggccc tctagatgca
     5821 tgctcgagcg gccgcttagc aactgcagct ttctgccgag gccttggccc ggtcattctt
     5881 gtccagtttg ataggttcag gaaattcgtt gtacagctcc acctccgttt cctgcttaag
     5941 tgcattccgt gcaatcgtct ggaacgcctg ctccacgttg atggcctcct tggcactggt
     6001 ctcaaagtag ggaatgttgt ttttgctgta gcaccaggcc tgtgcccgct ttgtggccac
     6061 ttgtctgttt tcgaggtcaa tcttgtttcc caacacaaca aatgggaagt tttcaggatc
     6121 tcggggactg gcctggatga gaaactcatc tctccagcta tctagggttt tgaatgtgtt
     6181 gggggcagtc acatcaaata ccagaacgca gcagtctgca cctctgtaga aggccacacc
     6241 gagagactgg aaccgttcca gtcctgctgt gtcccatatc tgcattgtga ctagcctgtc
     6301 atccaccatc acctccttgg tcagaaagtc agctcctatt gtggctttgt actgattgct
     6361 gaatttctta ttcacatact ggttcatgag tgatgtcttc ccgactccag aatctcccag
     6421 gatgataacc ttcagcaaca ctttcttcct agaggtacca gcgtaatccg gaacatcgta
     6481 tgggtaagcg taatccggaa catcgtatgg gtacatggat cccttgtaca gctcgtccat
     6541 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     6601 gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac
     6661 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc
     6721 ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat
//
