LOCUS       sequence-314524-        7933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ATG8 fused at the N terminus to GFP under the control of 
            the endogenous promoter , NAT selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7933)
  TITLE     Yeast viruses
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7933)
  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: 
            "Unpublished"
COMMENT     Sequence Label: GFP-ATG8(416)/GFP-AUT7(416)
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             652..1359
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(676..704)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1299..1319
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     gene            complement(1951..3070)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(1951..2148)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2154..2726)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(2727..3070)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(3198..3216)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3237..3253)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3237..3253)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3250..3272)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3261..3277
                     /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(3285..3291,3292..3309,3310..3315))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3330..3351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3468..3485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3639..4227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3719..3738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4398..5189,5190..5258))
                     /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     5021..5040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5259..5363)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5400..5903
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5944..5962
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6000..6022)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6122..6141
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6165..6380
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             6381..7184
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(7315..7770)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7452..7473)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7664..7683
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7896..7918
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7910..7927
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7911..7927
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattggag ctccaccgcg gtggcggccg ctctagaact
       61 agtaggtctc gcaagagagc tggtcaacag aatccaaaaa ttgagaaaga agtgtggttt
      121 ggaagccacc gacgatgttt tagtggagta cgaattagtt aaagatacta tcgactttga
      181 agccattgtc aaagaacatt ttgatatgtt aagcaagacc tgtagatccg acattgccaa
      241 atatgacggc tcaaagacag acccaattgg tgatgaagaa caatctatta atgacaccat
      301 tttcaaatta aaagtgttca aattatgaaa acaactcata taaatacgta caaatttttc
      361 tctactcgaa gtgatataga tgtatatgtg taagtttacg tttaagttag agtcatgtaa
      421 tgctaactgt ctccaccgat aatgttgtat aatacccgtg aaatcatagc acatgatata
      481 tcatcacccg gaggccggtt attttcggcg gcggcaaaaa tatttggtat aattatggaa
      541 atacaaaaag gggaaccatt aaaggttgag gaggggattg ataagagaat ctaataattg
      601 taaagttgag aaaatcataa taaaataatt actagagaca tgggatccgg taaaggagaa
      661 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
      721 aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa
      781 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcact
      841 tatggtgttc aatgcttttc aagataccca gatcatatga aacggcatga ctttttcaag
      901 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
      961 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
     1021 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
     1081 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
     1141 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
     1201 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
     1261 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
     1321 gctgctggga ttacacatgg catggatgaa ctatacaaag gatccaagtc tacatttaag
     1381 tctgaatatc catttgaaaa aaggaaggcg gagtcggaga ggattgctga caggttcaag
     1441 aataggatac ctgtgatttg cgaaaaagct gaaaagtcag atattccaga gattgataag
     1501 cgtaaatatc tagttcctgc tgaccttacc gtagggcaat ttgtttatgt tataagaaag
     1561 agaattatgc taccccctga gaaggccatc ttcatttttg tcaatgatac tttgccacct
     1621 actgcggcgt tgatgtctgc catatatcaa gaacacaagg ataaggacgg gtttttgtat
     1681 gtcacttact caggagaaaa tacatttggc aggtagtctt ttatatgaaa agaaatgaag
     1741 cgttaacatc taaaatcgaa attataggga ctcattagcc atctattgtt ccagtataag
     1801 aatgtttaca taaaaaattc gaaaaaaaaa gataaaataa gggaaaaata tatatatatt
     1861 cattaagaag ttttgtacac aacggagcca agatttcgaa ttcgagctcg ttttcgacac
     1921 tggatggcgg cgttagtatc gaatcgacag cagtatagcg accagcattc acatacgatt
     1981 gacgcatgat attactttct gcgcacttaa cttcgcatct gggcagatga tgtcgaggcg
     2041 aaaaaaaata taaatcacgc taacatttga ttaaaataga acaactacaa tataaaaaaa
     2101 ctatacaaat gacaagttct tgaaaacaag aatcttttta ttgtcagtac tgattagggg
     2161 cagggcatgc tcatgtagag cgcctgctcg ccgtccgagg cggtgccgtc gtacagggcg
     2221 gtgtccaggc cgcagagggt gaaccccatc cgccggtacg cgtggatcgc cggtgcgttg
     2281 acgttggtga cctccagcca gaggtgcccg gcgccccgct cgcgggcgaa ctccgtcgcg
     2341 agccccatca acgcgcgccc gaccccgtgc ccccggtgct ccggggcgac ctcgatgtcc
     2401 tcgacggtca gccggcggtt ccagccggag tacgagacga ccacgaagcc cgccaggtcg
     2461 ccgtcgtccc cgtacgcgac gaacgtccgg gagtccgggt cgccgtcctc cccgtcgtcc
     2521 gattcgtcgt ccgattcgtc gtcggggaac accttggtca ggggcgggtc caccggcacc
     2581 tcccgcaggg tgaagccgtc cccggtggcg gtgacgcgga agacggtgtc ggtggtgaag
     2641 gacccatcca gtgcctcgat ggcctcggcg tcccccggga cactggtgcg gtaccggtaa
     2701 gccgtgtcgt caagagtggt acccatggtt gtttatgttc ggatgtgatg tgagaactgt
     2761 atcctagcaa gattttaaaa ggaagtatat gaaagaagaa cctcagtggc aaatcctaac
     2821 cttttatatt tctctacagg ggcgcggcgt ggggacaatt caacgcgtct gtgaggggag
     2881 cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt tttgcttcgc gccgtgcggc
     2941 catcaaaatg tatggatgca aatgattata catggggatg tatgggctaa atgtacgggc
     3001 gacagtcaca tcatgcccct gagctgcgca cgtcaagact gtcaaggagg gtattctggg
     3061 cctccatgtc gctggccggg tgacccggcg gggacgaggc aagctaaaca gatctggcgc
     3121 gccttaatta acccggggat ccgtcgacct gcagcgtacg aagcttcagc tggcggccgc
     3181 tctagcagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
     3241 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3301 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3361 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3421 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3481 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3541 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3601 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3661 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3721 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3781 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3841 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3901 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3961 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4021 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4081 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4141 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4201 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4261 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4321 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4381 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4441 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4501 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4561 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4621 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4681 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4741 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4801 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4861 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4921 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4981 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5041 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5101 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5161 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5221 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5281 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5341 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtccttttca
     5401 tcacgtgcta taaaaataat tataatttaa attttttaat ataaatatat aaattaaaaa
     5461 tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg ttttccgaag atgtaaaaga
     5521 ctctaggggg atcgccaaca aatactacct tttatcttgc tcttcctgct ctcaggtatt
     5581 aatgccgaat tgtttcatct tgtctgtgta gaagaccaca cacgaaaatc ctgtgatttt
     5641 acattttact tatcgttaat cgaatgtata tctatttaat ctgcttttct tgtctaataa
     5701 atatatatgt aaagtacgct ttttgttgaa attttttaaa cctttgttta tttttttttc
     5761 ttcattccgt aactcttcta ccttctttat ttactttcta aaatccaaat acaaaacata
     5821 aaaataaata aacacagagt aaattcccaa attattccat cattaaaaga tacgaggcgc
     5881 gtgtaagtta caggcaagcg atccgtctaa gaaaccatta ttatcatgac attaacctat
     5941 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     6001 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     6061 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     6121 gcggcatcag agcagattgt actgagagtg caccatacca ccttttcaat tcatcatttt
     6181 ttttttattc ttttttttga tttcggtttc cttgaaattt ttttgattcg gtaatctccg
     6241 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta
     6301 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct
     6361 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc
     6421 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg
     6481 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca
     6541 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag
     6601 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat
     6661 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag
     6721 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga
     6781 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt
     6841 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga
     6901 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag
     6961 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg
     7021 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg
     7081 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag
     7141 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag
     7201 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac
     7261 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     7321 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     7381 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     7441 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     7501 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     7561 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     7621 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     7681 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7741 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     7801 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     7861 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     7921 ggccagtgaa ttg
//
