LOCUS       sequence-256237-        7477 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 7477)
  AUTHORS   Alberti S, Gitler AD, Lindquist S
  TITLE     A suite of Gateway cloning vectors for high-throughput genetic 
            analysis in Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2007 Oct . 24(10):913-9.
  PUBMED    17583893
REFERENCE   2  (bases 1 to 7477)
  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: "Yeast. 2007
            Oct . 24(10):913-9."
COMMENT     Sequence Label: pAG416GPD-ccdB
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2754..2774
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2799..3442
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3336..3359
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3448..3464
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3448..3464
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3472..3596
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3621..3626,3627..3644,3645..3651)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3705..4364
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3771..3790)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4706..5011
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4890..4909
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5052..5176)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5209..5225)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5210..5226)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5228..5475
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5244..5262)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5493..5512)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5494..5512)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5522..5539)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5522..5538)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5531..5553)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5679..6134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5766..5785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5976..5997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6265..7068)
                     /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"
     promoter        complement(7069..7284)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7308..7327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7427..7449
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tcagtttatc attatcaata ctcgccattt
     2821 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt
     2881 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
     2941 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
     3001 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt
     3061 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc
     3121 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat
     3181 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
     3241 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa
     3301 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
     3361 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta
     3421 aaacaccaga acttagtttc gacggattct agaactagtg gatcccccat cacaagtttg
     3481 tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     3541 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg
     3601 ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt
     3661 tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     3721 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     3781 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     3841 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     3901 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     3961 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     4021 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     4081 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     4141 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     4201 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     4261 cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta
     4321 atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct
     4381 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
     4441 atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac
     4501 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     4561 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa
     4621 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt
     4681 gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag
     4741 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     4801 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     4861 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     4921 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     4981 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc
     5041 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt
     5101 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt
     5161 tcttgtacaa agtggtgatg ggctgcagga attcgatatc aagcttatcg ataccgtcga
     5221 cctcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
     5281 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     5341 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
     5401 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
     5461 aaggctttaa tttgcggccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     5521 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     5581 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     5641 gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt
     5701 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5761 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5821 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5881 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5941 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6001 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6061 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6121 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6181 cgcatagggt aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca
     6241 tgcatttact tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc
     6301 ccagcctgct tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag
     6361 tcctcttcca acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata
     6421 ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca
     6481 atcgtaacct tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc
     6541 tttgtcgctc ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc
     6601 cttgcatgac aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc
     6661 cgcctgcttc aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc
     6721 tgcccattct gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat
     6781 gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag
     6841 cggcttaact gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa
     6901 acaaattttg ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc
     6961 caatgaagca cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac
     7021 aggactagga tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg
     7081 tttcctgcag gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc
     7141 ttcaacacta catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc
     7201 cttctgttcg gagattaccg aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga
     7261 ataaaaaaaa aatgatgaat tgaaaaggtg gtatggtgca ctctcagtac aatctgctct
     7321 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     7381 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     7441 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
