LOCUS       sequence-46353-e        7194 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 7194)
  AUTHORS   Zhu Y, Xu J, Heinemann SF
  TITLE     Two pathways of synaptic vesicle retrieval revealed by 
            single-vesicle imaging.
  JOURNAL   Neuron. 2009 Feb 12;61(3):397-411.
  PUBMED    19217377
REFERENCE   2  (bases 1 to 7194)
  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: "Neuron."; 
            date: "2009-02-12"; volume: "61(3)"; pages: "397-411"
COMMENT     Sequence Label: pcDNA3-SypHluorin 1x (S1x)
FEATURES             Location/Qualifiers
     source          1..7194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      936..945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1491..1500
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1497..2213
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Miesenbock et al., 1998)"
                     /label=ecliptic pHluorin
                     /note="responds to a drop in pH with a gradual loss 
                     (eclipsing) of excitation at 475 nm, accompanied by a 
                     reduction of excitation at 395 nm"
                     /translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1530..1558)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2153..2173
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2637..2663
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2678..2707
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2723..2740
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2784..2801)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2790..3014
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3060..3488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3147..3166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3357..3378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3497..3517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3502..3832
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3683..3818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3745..3764
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3899..4693
                     /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(3953..3972)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4563..4582
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4867..4988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4904..4923)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4958..4977
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5037..5053)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5037..5053)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5050..5072)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5061..5077
                     /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(5085..5091,5092..5109,5110..5115))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5130..5151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5268..5285)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5439..6027)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5519..5538)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6198..6989,6990..7058))
                     /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     6821..6840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7059..7163)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagctca agcttgccac catggacgtg gtgaatcagc
      961 tggtggctgg gggtcagttc cgggtggtca aggagcccct tggcttcgtg aaggtgctgc
     1021 agtgggtctt tgccatcttc gcctttgcta cgtgcggcag ctacaccgga gagcttcggc
     1081 tgagcgtgga gtgtgccaac aagacggaga gtgccctcaa catcgaagtc gaatttgagt
     1141 acccattcag gctgcaccaa gtgtactttg atgcaccctc ctgcgttaaa gggggcacta
     1201 ccaagatctt cctagttggt gactactcct cctcgggtga attctttgtc accgtggctg
     1261 tgtttgcctt cctctactcc atgggggccc tggccaccta catcttcctg cagaacaagt
     1321 accgagagaa caacaaaggg ccaatgatgg acttcctggc cacagcagtg ttcgctttca
     1381 tgtggctagt tagctcatcc gcctgggcca aaggcctgtc cgatgtgaag atggccactg
     1441 acccagagaa cattatcaag gagatgccta tgtgccgcca gacagatcta gccaccatgg
     1501 gaagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
     1561 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
     1621 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
     1681 tcactacttt ctcttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc
     1741 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
     1801 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
     1861 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
     1921 tggaatacaa ctataacgat caccaggtgt acatcatggc agacaaacaa aagaatggaa
     1981 tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg aggcgttcaa ctagcagacc
     2041 attatcaaca aaatactcca attggcgatg ggcccgtcct tttaccagac aaccattacc
     2101 tgtttacaac ttctactctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
     2161 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaaaccggtg
     2221 gagctcgaat cacatgcaag gaactgaggg accctgtgac ttcaggactc aacacctcgg
     2281 tggtgtttgg cttcctgaac ctggtgctct gggttggcaa cctatggttc gtgttcaagg
     2341 agacaggctg ggccgcccca ttcatgcgcg cacctccagg cgccccagaa aagcaaccag
     2401 ctcctggcga tgcctacggc gatgcgggct atgggcaggg ccccggaggc tatgggcccc
     2461 aggactccta cgggcctcag ggtggttatc aacccgatta cgggcagcca gccagcggcg
     2521 gtggcggtgg ctacgggcct cagggcgact atgggcagca aggctacggc caacagggtg
     2581 cgcccacctc cttctccaat cagatgtcgg atccgaagct tcggtaccaa gctgcctacc
     2641 cttacgatgt gcccgattac gcctgatagc ttacgtagaa caaaaactca tctcagaaga
     2701 ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttaaa cggtctccag
     2761 cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2821 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2881 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2941 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3001 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3061 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3121 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3181 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     3241 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3301 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3361 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3421 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3481 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3541 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3601 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3661 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3721 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3781 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3841 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3901 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3961 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4021 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4081 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4141 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4201 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4261 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4321 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4381 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4441 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4501 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4561 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4621 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4681 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4741 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4801 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4861 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4921 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4981 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5041 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5101 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5161 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5221 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5281 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5341 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5401 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5461 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5521 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5581 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     5641 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5701 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5761 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5821 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5881 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5941 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6001 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6061 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6121 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6181 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6241 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6301 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6361 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6421 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6481 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6541 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6601 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6661 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6721 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6781 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6841 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6901 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6961 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7021 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7081 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7141 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
