LOCUS       sequence-90653-e        7859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multicopy with clonNat resistance marker for single Gateway 
            insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7859)
  TITLE     Fluorescent X-QTL
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7859)
  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            "Unpublished"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: p42Nat 1-F GW
FEATURES             Location/Qualifiers
     source          1..7859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /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)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(3486..3491,3492..3509,3510..3516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /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     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     3640..3656
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3640..3656
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3681..3805
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3830..3835,3836..3853,3854..3860)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3914..4573
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3980..3999)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4918..5223
                     /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     5102..5121
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5264..5388)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5437..5453)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5438..5454)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5456..5703
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5472..5490)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5721..5740)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5722..5740)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5750..5767)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5750..5766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5759..5781)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5911..6366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5998..6017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6208..6229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     gene            complement(6496..7615)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(6496..6693)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(6699..7271)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(7272..7615)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(7690..7709)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7809..7831
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagaactag tggatccccc
     3661 gaactagtgg atcccccatc acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg
     3721 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     3781 aaacacaaca tatccagtca ctatggcggc cgcattaggc accccaggct ttacacttta
     3841 tgcttccggc tcgtataatg tgtggatttt gagttaggat ccgtcgagat tttcaggagc
     3901 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     3961 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4021 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4081 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc
     4141 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     4201 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     4261 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     4321 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     4381 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     4441 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtctg
     4501 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     4561 gggcggggcg taaacgccgc gtggatccgg cttactaaaa gccagataac agtatgcgta
     4621 tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc
     4681 aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag
     4741 ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg
     4801 aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg
     4861 tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg
     4921 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     4981 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     5041 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     5101 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
     5161 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     5221 taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg actggatatg
     5281 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
     5341 atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtga tgggctgcag
     5401 gaattcgggg ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagtcatg
     5461 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     5521 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     5581 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     5641 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     5701 tgcggccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     5761 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5821 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5881 agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca ttaagcgcgg
     5941 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     6001 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     6061 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     6121 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6181 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6241 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6301 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     6361 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatagc
     6421 tagtggatct gatatcatcg atgaattcga gctcgttttc gacactggat ggcggcgtta
     6481 gtatcgaatc gacagcagta tagcgaccag cattcacata cgattgacgc atgatattac
     6541 tttctgcgca cttaacttcg catctgggca gatgatgtcg aggcgaaaaa aaatataaat
     6601 cacgctaaca tttgattaaa atagaacaac tacaatataa aaaaactata caaatgacaa
     6661 gttcttgaaa acaagaatct ttttattgtc agtactgatt aggggcaggg catgctcatg
     6721 tagagcgcct gctcgccgtc cgaggcggtg ccgtcgtaca gggcggtgtc caggccgcag
     6781 agggtgaacc ccatccgccg gtacgcgtgg atcgccggtg cgttgacgtt ggtgacctcc
     6841 agccagaggt gcccggcgcc ccgctcgcgg gcgaactccg tcgcgagccc catcaacgcg
     6901 cgcccgaccc cgtgcccccg gtgctccggg gcgacctcga tgtcctcgac ggtcagccgg
     6961 cggttccagc cggagtacga gacgaccacg aagcccgcca ggtcgccgtc gtccccgtac
     7021 gcgacgaacg tccgggagtc cgggtcgccg tcctccccgt cgtccgattc gtcgtccgat
     7081 tcgtcgtcgg ggaacacctt ggtcaggggc gggtccaccg gcacctcccg cagggtgaag
     7141 ccgtccccgg tggcggtgac gcggaagacg gtgtcggtgg tgaaggaccc atccagtgcc
     7201 tcgatggcct cggcgtcccc cgggacactg gtgcggtacc ggtaagccgt gtcgtcaaga
     7261 gtggtaccca tggttgttta tgttcggatg tgatgtgaga actgtatcct agcaagattt
     7321 taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt atatttctct
     7381 acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt ccctgctcgc
     7441 aggtctgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca aaatgtatgg
     7501 atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag tcacatcatg
     7561 cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc atgtcgctgg
     7621 ccgggtgacc cggcggggac aaggcaagct aaacagatct tgaaaagctg tggtatggtg
     7681 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     7741 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     7801 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
