LOCUS       sequence-90662-e        8299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multicopy with clonNat resistance marker for triple Gateway 
            insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8299)
  TITLE     Fluorescent X-QTL
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8299)
  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            "Unpublished"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 3-F GW
FEATURES             Location/Qualifiers
     source          1..8299
                     /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    3677..3800
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4148..4453)
                     /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     complement(4250..4269)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(4773..5456)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     5371..5390
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5457..5559)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(5705..5828)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5876..5892)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5877..5893)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5895..6142
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5911..5929)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6160..6179)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6161..6179)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6189..6205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6198..6220)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6350..6805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6437..6456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6647..6668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     gene            complement(6935..8054)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(6935..7132)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(7138..7710)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(7711..8054)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(8130..8149)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8249..8271
                     /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 gattacgcca agctatcaac tttgtataga aaagttgaac gagaaacgta aaatgatata
     3721 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     3781 caacatatcc agtcactatg gtcgaccgac agccttccaa atgttcttct caaacggaat
     3841 cgtcgtatcc agcctactcg ctattgtcct caatgccgta ttaaatcata aaaagaaata
     3901 agaaaaagag gtgcgagcct cttttttgtg tgacaaaata aaaacatcta cctattcata
     3961 tacgctagtg tcatagtcct gaaaatcatc tgcatcaaga acaatttcac aactcttata
     4021 cttttctctt acaagtcgtt cggcttcatc tggattttca gcctctatac ttactaaacg
     4081 tgataaagtt tctgtaattt ctactgtatc gacctgcaga ctggctgtgt ataagggagc
     4141 ctgacattta tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt
     4201 ggctgagatc agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg
     4261 tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt
     4321 tatctgacag cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa
     4381 taatatcact ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa
     4441 ccttaaactg catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata
     4501 aaccgggcga cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga
     4561 cgacgggctt cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg
     4621 ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat
     4681 acctcttttt gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc
     4741 agcgcgcaaa tacgcatact gttatctggc ttttagtaag ccggatccac gcggcgttta
     4801 cgccccccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     4861 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     4921 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     4981 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
     5041 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     5101 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     5161 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     5221 tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     5281 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     5341 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     5401 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     5461 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     5521 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
     5581 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
     5641 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttacgg
     5701 ccgccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     5761 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     5821 catagttgat aattcactgg ccgtcggggc tgcaggaatt cgatatcaag cttatcgata
     5881 ccgtcgacct cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     5941 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     6001 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     6061 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     6121 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
     6181 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     6241 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     6301 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt
     6361 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6421 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6481 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6541 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6601 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6661 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     6721 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     6781 aacaaaatat taacgtttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     6841 gtatttcaca ccgcatagct agtggatctg atatcatcga tgaattcgag ctcgttttcg
     6901 acactggatg gcggcgttag tatcgaatcg acagcagtat agcgaccagc attcacatac
     6961 gattgacgca tgatattact ttctgcgcac ttaacttcgc atctgggcag atgatgtcga
     7021 ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa tagaacaact acaatataaa
     7081 aaaactatac aaatgacaag ttcttgaaaa caagaatctt tttattgtca gtactgatta
     7141 ggggcagggc atgctcatgt agagcgcctg ctcgccgtcc gaggcggtgc cgtcgtacag
     7201 ggcggtgtcc aggccgcaga gggtgaaccc catccgccgg tacgcgtgga tcgccggtgc
     7261 gttgacgttg gtgacctcca gccagaggtg cccggcgccc cgctcgcggg cgaactccgt
     7321 cgcgagcccc atcaacgcgc gcccgacccc gtgcccccgg tgctccgggg cgacctcgat
     7381 gtcctcgacg gtcagccggc ggttccagcc ggagtacgag acgaccacga agcccgccag
     7441 gtcgccgtcg tccccgtacg cgacgaacgt ccgggagtcc gggtcgccgt cctccccgtc
     7501 gtccgattcg tcgtccgatt cgtcgtcggg gaacaccttg gtcaggggcg ggtccaccgg
     7561 cacctcccgc agggtgaagc cgtccccggt ggcggtgacg cggaagacgg tgtcggtggt
     7621 gaaggaccca tccagtgcct cgatggcctc ggcgtccccc gggacactgg tgcggtaccg
     7681 gtaagccgtg tcgtcaagag tggtacccat ggttgtttat gttcggatgt gatgtgagaa
     7741 ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag tggcaaatcc
     7801 taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc gtctgtgagg
     7861 ggagcgtttc cctgctcgca ggtctgcagc gaggagccgt aatttttgct tcgcgccgtg
     7921 cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg ctaaatgtac
     7981 gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag gagggtattc
     8041 tgggcctcca tgtcgctggc cgggtgaccc ggcggggaca aggcaagcta aacaagatct
     8101 tgaaaagctg tggtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     8161 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     8221 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     8281 tcatcaccga aacgcgcga
//
