LOCUS       sequence-436829-        6641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of YeiN in E. coli BL21_DE3 (pLys) or similar.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  TITLE     Efficient synthetic access to stable isotope labelled pseudouridine 
            phosphoramidites for RNA NMR spectroscopy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6641)
  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            "Unpublished"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: pet15b _yein
FEATURES             Location/Qualifiers
     source          1..6641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        173..250
                     /gene="lacI"
                     /label=lacI promoter
     CDS             251..1333
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(270..289)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2645..2836
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2830..2852)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2938..3077
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2993..3012
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3092..3109)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3263..3851)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3343..3362)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4022..4813,4814..4882))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4645..4664
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4883..4987)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5055..5073
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5100..5105,5106..5122,5123..5128)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(5303..5350)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5346..5364
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6362..6379)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(6389..6406)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6425..6447
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6462..6486
                     /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     complement(6486..6505)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6487..6505)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6589..6608)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
       61 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
      121 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
      181 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
      241 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
      301 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
      361 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
      421 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
      481 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
      541 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
      601 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
      661 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
      721 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
      781 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
      841 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
      901 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
      961 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     1021 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     1081 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     1141 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     1201 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     1261 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     1321 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     1381 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     1441 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     1501 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     1561 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     1621 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     1681 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     1741 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     1801 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
     1861 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
     1921 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     1981 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     2041 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc
     2101 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     2161 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     2221 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     2281 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     2341 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     2401 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     2461 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     2521 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     2581 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     2641 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     2701 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     2761 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     2821 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2881 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     2941 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3001 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     3061 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3121 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3181 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3241 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3301 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3361 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3421 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3481 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3541 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3601 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3661 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     3721 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3781 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3841 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3901 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3961 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4021 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4081 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4141 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4201 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4261 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4321 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4381 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4441 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4501 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4561 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4621 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4681 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4741 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4801 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4861 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4921 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4981 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5041 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttctcatgtt
     5101 tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa ttgctaacgc
     5161 agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc
     5221 ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc
     5281 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     5341 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
     5401 tagcagccgg atcctcgagt taacccgcga gacgctgata ttctttggca atttcgctcg
     5461 ccagaatggc gttgttgaac accagctgga tgttggattt caggctgtca ccgccggtca
     5521 gttcagcaac gcgcgccagc aggaatggcg tactttcttt accaatgact ccctgagctt
     5581 cagcttcagc taccgcctga tcgatcgccg cattgatagt gtgttccggc atagcaaact
     5641 gttccgggat cgggttcgct accacgaggc caccgttcag accgctttgc catttcacca
     5701 ccattgcacg ggcaatttcg ctggcgctgt cgagacgaat gctgacgtca aacgggctgg
     5761 tgcggcagaa aaacgcaggc agcgctttag tctgatagcc aattaacggc acaccgaagg
     5821 tttctaaata ctcagtggtt aatcctaaat cgagaataga tttcgccccg gcacaaacaa
     5881 cggtgacatt agtatttgcc agttcttgca aatcggcaga aatatcgaag gtatgttccg
     5941 ccccgcgatg cacaccacca attcccccgg tggcaaatac tttaattccg gcaagcgccg
     6001 caataatcat cgttgaagcc acagtggttg cgccattttt tccggcggca acaacaaaag
     6061 gtaaatcgcg acgactaact ttggtcacgt tatgcccttc acgacccagt aattcaattt
     6121 cttctttgct taaccccact ttcatcacgc cgccaataat ggcgatcgtg gcaggtacag
     6181 cgccctgttt acgaatagtt tcttcaactt caattgcggt ctgggcattt tgtgggaacg
     6241 gcatcccgtg agaaataatg gtcgattcca gcgccacaac cggttttttg ttttttaaag
     6301 cgtcctgcac ttccggggaa atttgtaata attcagggga aatttttaat tcagacatat
     6361 ggctgccgcg cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatgg
     6421 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     6481 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
     6541 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     6601 acatcaccga tggggaagat cgggctcgcc acttcgggct c
//
