LOCUS       sequence-405511-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcribes a glmZ' (1-146)-gfp fusion. gfp can be replaced via 
            AgeI/XbaI-sites with gene of interest to release its RNA with a 5' 
            monophosphate upon cleavage..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  AUTHORS   Schilder A, Gorke B
  TITLE     Role of the 5' end phosphorylation state for small RNA stability and
            target RNA regulation in bacteria.
  JOURNAL   Nucleic Acids Res. 2023 Mar 29:gkad226. doi: 10.1093/nar/gkad226.
  PUBMED    36987877
REFERENCE   2  (bases 1 to 6379)
  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: "Nucleic 
            Acids Res. 2023 Mar 29:gkad226. doi: 10.1093/nar/gkad226."
COMMENT     Sequence Label: pYG215
FEATURES             Location/Qualifiers
     source          1..6379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..153
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      820..1042
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1090..2040
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     promoter        2146..2223
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             2224..3306
                     /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(2243..2262)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    3389..3405
                     /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)."
     CDS             3598..4311
                     /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="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3628..3656)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4251..4271
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        join(4360..4365,4366..4382,4383..4388)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4436..5626
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(4788..4807)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(join(5939..6379,1..219))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
       61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
      121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
      181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
      241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
      301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
      361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
      421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt
      481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg
      541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgcctgtag tgccatttac
      601 ccccattcac tgccagagcc gtgagcgcag cgaactgaat gtcacgaaaa agacagcgac
      661 tcaggtgcct gatggtcgga gacaaaagga atattcagcg atttgcccga gcttgcgagg
      721 gtgctactta agcctttagg gttttaaggt ctgttttgta gaggagcaaa cagcgtttgc
      781 gacatccttt tgtaatactg cggaactgac taaagtagtg agttatacac agggctggga
      841 tctattcttt ttatcttttt ttattctttc tttattctat aaattataac cacttgaata
      901 taaacaaaaa aaacacacaa aggtctagcg gaatttacag agggtctagc agaatttaca
      961 agttttccag caaaggtcta gcagaattta cagataccca caactcaaag gaaaaggact
     1021 agtaattatc attgactagc ccatctcaat tggtatagtg attaaaatca cctagaccaa
     1081 ttgagatgta tgtctgaatt agttgttttc aaagcaaatg aactagcgat tagtcgctat
     1141 gacttaacgg agcatgaaac caagctaatt ttatgctgtg tggcactact caaccccacg
     1201 attgaaaacc ctacaaggaa agaacggacg gtatcgttca cttataacca atacgctcag
     1261 atgatgaaca tcagtaggga aaatgcttat ggtgtattag ctaaagcaac cagagagctg
     1321 atgacgagaa ctgtggaaat caggaatcct ttggttaaag gctttgagat tttccagtgg
     1381 acaaactatg ccaagttctc aagcgaaaaa ttagaattag tttttagtga agagatattg
     1441 ccttatcttt tccagttaaa aaaattcata aaatataatc tggaacatgt taagtctttt
     1501 gaaaacaaat actctatgag gatttatgag tggttattaa aagaactaac acaaaagaaa
     1561 actcacaagg caaatataga gattagcctt gatgaattta agttcatgtt aatgcttgaa
     1621 aataactacc atgagtttaa aaggcttaac caatgggttt tgaaaccaat aagtaaagat
     1681 ttaaacactt acagcaatat gaaattggtg gttgataagc gaggccgccc gactgatacg
     1741 ttgattttcc aagttgaact agatagacaa atggatctcg taaccgaact tgagaacaac
     1801 cagataaaaa tgaatggtga caaaatacca acaaccatta catcagattc ctacctacat
     1861 aacggactaa gaaaaacact acacgatgct ttaactgcaa aaattcagct caccagtttt
     1921 gaggcaaaat ttttgagtga catgcaaagt aagtatgatc tcaatggttc gttctcatgg
     1981 ctcacgcaaa aacaacgaac cacactagag aacatactgg ctaaatacgg aaggatctga
     2041 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
     2101 aactgttcac cgttacatat caaagggaaa actgtccata tgcccgacac catcgaatgg
     2161 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     2221 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     2281 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     2341 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     2401 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     2461 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     2521 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     2581 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     2641 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     2701 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     2761 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     2821 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     2881 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     2941 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     3001 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     3061 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     3121 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     3181 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     3241 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     3301 cagtgagcgc aacgcaatta atgtaagtta gctcaaatat tctgaaatga gctgttctgc
     3361 agataaatgt gagcggataa cattgacatt gtgagcggat aacaagatac tgacgtcgta
     3421 gatgctcatt ccatctctta tgttcgcctt agtgcctcat aaactccgga atgacgcaga
     3481 gccgtttacg gtgcttatcg tccactgaca gatgtcgctt atgcctcatc agacaccatg
     3541 gacacaacgt tgagtgaagc accggtcaaa gtcggtgaca gataacagga gtaagtaatg
     3601 cgtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat
     3661 gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa
     3721 cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc
     3781 actactttcg gttatggtgt tcaatgcttt gcgagatacc cagatcatat gaaacagcat
     3841 gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa
     3901 gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat
     3961 agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg
     4021 gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc
     4081 aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat
     4141 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg
     4201 tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt
     4261 gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa ataatctaga
     4321 acgctcggtt gccgccgggc gttttttgaa ttctcatgtt tgacagctta tcatcgataa
     4381 gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa
     4441 atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg
     4501 cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg acagcatcgc
     4561 cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat gcgcacccgt
     4621 tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt cgctacttgg
     4681 agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggatcc tctacgccgg
     4741 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     4801 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     4861 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     4921 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     4981 ggagtcgcat aagggagagc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc
     5041 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     5101 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     5161 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     5221 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     5281 cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat
     5341 ggccttcccc attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc
     5401 catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc
     5461 tcttaccagc ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc
     5521 ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct
     5581 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     5641 cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact
     5701 gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc
     5761 cgcacgcggc gcatcccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     5821 cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc cgcttattat
     5881 cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct taaaaaaatt
     5941 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     6001 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     6061 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     6121 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     6181 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     6241 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     6301 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     6361 cgtctttcat tgccatacg
//
