LOCUS       sequence-217452-        4865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Selection for in-frame fusion expression constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4865)
  AUTHORS   McGeachy AM, Meacham ZA, Ingolia NT
  TITLE     An Accessible Continuous-Culture Turbidostat for Pooled Analysis of 
            Complex Libraries.
  JOURNAL   ACS Synth Biol. 2019 Apr 19;8(4):844-856. doi: 
            10.1021/acssynbio.8b00529. Epub 2019 Apr 2.
  PUBMED    30908907
REFERENCE   2  (bases 1 to 4865)
  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"; doi: 
            "10.1021/acssynbio.8b00529"; journalName: "ACS Synth Biol"; date: 
            "2019-04-19- 19"; volume: "8"; issue: "4"; pages: "844-856"
COMMENT     Sequence Label: pPGK1-(AscI)-lambda-N-T2A-SpHIS5 (pNTI347)
FEATURES             Location/Qualifiers
     source          1..4865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..181
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(219..241)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     341..360
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        441..459
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     441..458
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    505..1008
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             1885..1947
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="DAQTRRRERRAEKQAQWKAAN"
     CDS             1948..2001
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2002..2652
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="GRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHTNQK
                     GEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQA
                     MSNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLYS
                     FSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2658..2855
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2959..2978)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2960..2978)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3065..3082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3236..3824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3316..3335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3995..4786,4787..4855))
                     /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     4618..4637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4856..4865,1..95))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
      121 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
      181 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      241 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      301 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      361 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
      421 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
      481 tcgtacgctg caggggtcct tttcatcacg tgctataaaa ataattataa tttaaatttt
      541 ttaatataaa tatataaatt aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt
      601 ttttgttttc cgaagatgta aaagactcta gggggatcgc caacaaatac taccttttat
      661 cttgctcttc ctgctctcag gtattaatgc cgaattgttt catcttgtct gtgtagaaga
      721 ccacacacga aaatcctgtg attttacatt ttacttatcg ttaatcgaat gtatatctat
      781 ttaatctgct tttcttgtct aataaatata tatgtaaagt acgctttttg ttgaaatttt
      841 ttaaaccttt gtttattttt ttttcttcat tccgtaactc ttctaccttc tttatttact
      901 ttctaaaatc caaatacaaa acataaaaat aaataaacac agagtaaatt cccaaattat
      961 tccatcatta aaagatacga ggcgcgtgta agttacaggc aagcgatccg tccggatcct
     1021 gtagccctag acttgatagc catgacttca actcaagacg cacagatatt ataacatctg
     1081 cataataggc atttgcaaga attactcgtg agtaaggaaa gagtgaggaa ctatcgcata
     1141 cctgcattta aagatgccga tttgggcgcg aatcctttat tttggcttca ccctcatact
     1201 attatcaggg ccagaaaaag gaagtgtttc cctccttctt gaattgatgt taccctcata
     1261 aagcacgtgg cctcttatcg agaaagaaat taccgtcgct cgtgatttgt ttgcaaaaag
     1321 aacaaaactg aaaaaaccca gacacgctcg acttcctgtc ttcctattga ttgcagcttc
     1381 caatttcgtc acacaacaag gtcctagcga cggctcacag gttttgtaac aagcaatcga
     1441 aggttctgga atggcgggaa agggtttagt accacatgct atgatgccca ctgtgatctc
     1501 cagagcaaag ttcgttcgat cgtactgtta ctctctctct ttcaaacaga attgtccgaa
     1561 tcgtgtgaca acaacagcct gttctcacac actcttttct tctaaccaag ggggtggttt
     1621 agtttagtag aacctcgtga aacttacatt tacatatata taaacttgca taaattggtc
     1681 aatgcaagaa atacatattt ggtcttttct aattcgtagt ttttcaagtt cttagatgct
     1741 ttctttttct cttttttaca gatcatcaag gaagtaatta tctacttttt acaacaaatt
     1801 aattaaaatg acactctttc cctacacgac gctcttccga tctggcgcgc cgatcggaag
     1861 agcacacgtc tgaactccag tcacgacgca caaacacgac gacgtgagcg tcgcgctgag
     1921 aaacaagctc aatggaaagc tgcaaacgaa ggtcgtggat cactgctaac gtgtggagat
     1981 gtcgaagaga atccaggtcc tggtaggagg gcttttgtag aaagaaatac gaacgaaacg
     2041 aaaatcagcg ttgccatcgc tttggacaaa gctcccttac ctgaagagtc gaattttatt
     2101 gatgaactta taacttccaa gcatacaaac caaaagggag aacaagtaat ccaagtagac
     2161 acgggaattg gattcttgga tcacatgtat catgcactgg ctaaacatgc aggctggagc
     2221 ttacgacttt actcaagagg tgatttaatc atcgatgatc atcacactgc agaagatact
     2281 gctattgcac ttggtattgc attcaagcag gctatgagta actttgccgg cgttaaaaga
     2341 tttggacatg cttattgtcc acttgacgaa gctctttcta gaagcgtagt tgacttgtcg
     2401 ggacggccct atgctgttat cgatttggga ttaaagcgtg aaaaggttgg ggaattgtcc
     2461 tgtgaaatga tccctcactt actatattcc ttttcggtag cagctggaat tactttgcat
     2521 gttacctgct tatatggtag taatgaccat catcgtgctg aaagcgcttt taaatctctg
     2581 gctgttgcca tgcgcgcggc tactagtctt actggaagtt ctgaagtccc aagcacgaag
     2641 ggagtgttgt aaagagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg
     2701 tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt
     2761 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     2821 gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc
     2881 atccagttta aacgagctcg aattcatcga tgatatcaga tccactagtg gcctatgcgg
     2941 ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc
     3001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3061 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3421 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3661 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4861 tcctt
//
