LOCUS       sequence-242696-        5810 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5810)
  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 5810)
  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: "Proc Natl 
            Acad Sci U S A. 2010 Nov 9. 107(45):19338-43."
COMMENT     Sequence Label: Flag-Rab5A
FEATURES             Location/Qualifiers
     source          1..5810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        472..675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     626..646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    677..695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    698..716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1513..1578
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1619..1636)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1625..1849
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1982..2001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2133..2180
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2188..3210
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2188..3210
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3340..3461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3377..3396)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3431..3450
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3510..3526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3510..3526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3523..3545)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3534..3550
                     /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(3558..3564,3565..3582,3583..3588))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3603..3624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3741..3758)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3912..4500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3992..4011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4671..5462,5463..5531))
                     /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     5294..5313
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5532..5636)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5711..5730)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
       61 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      661 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
      721 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      781 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
      841 ggtaccgagc tcggatccat ggctagtcga ggcgcaacaa gacccaacgg gccaaatact
      901 ggaaataaaa tatgccagtt caaactagta cttctgggag agtccgctgt tggcaaatca
      961 agcctagtgc ttcgttttgt gaaaggccaa tttcatgaat ttcaagagag taccattggg
     1021 gctgcttttc taacccaaac tgtatgtctt gatgacacta cagtaaagtt tgaaatatgg
     1081 gatacagctg gtcaagaacg ataccatagc ctagcaccaa tgtactacag aggagcacaa
     1141 gcagccatag ttgtatatga tatcacaaat gaggagtcct ttgcaagagc aaaaaattgg
     1201 gttaaagaac ttcagaggca agcaagtcct aacattgtaa tagctttatc gggaaacaag
     1261 gccgacctag caaataaaag agcagtagat ttccaggaag cacagtccta tgcagatgac
     1321 aatagtttat tattcatgga gacatccgct aaaacatcaa tgaatgtaaa tgaaatattc
     1381 atggcaatag ctaaaaaatt gccaaagaat gaaccacaaa atccaggagc aaattctgcc
     1441 agaggaagag gagtagacct taccgaaccc acacaaccaa ccaggaatca gtgttgtagt
     1501 aacggcggcc gcgactacaa agaccatgac ggtgattata aagatcatga catcgactac
     1561 aaggatgacg atgacaagtg actcgagtct agagggcccg tttaaacccg ctgatcagcc
     1621 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1681 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1741 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1801 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     1861 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     1921 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1981 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2041 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2101 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     2161 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     2221 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     2281 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     2341 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     2401 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     2461 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     2521 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     2581 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     2641 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     2701 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     2761 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     2821 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     2881 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     2941 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     3001 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     3061 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     3121 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     3181 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     3241 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3301 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3361 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3421 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     3481 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3541 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3601 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3661 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3721 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3781 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3841 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3901 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3961 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4021 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4081 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4141 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4201 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4261 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4321 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4381 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4441 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4501 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4561 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4621 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4681 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4741 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4801 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4861 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4921 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4981 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5041 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5101 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5161 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5221 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5281 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5341 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5401 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5461 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5521 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5581 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5641 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     5701 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     5761 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
//
