LOCUS       sequence-58062-e        6569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6569)
  AUTHORS   Hackel BJ, Kapila A, Wittrup KD
  TITLE     Picomolar affinity fibronectin domains engineered utilizing loop 
            length diversity, recursive mutagenesis, and loop shuffling.
  JOURNAL   J Mol Biol. 2008 Sep 19;381(5):1238-52. doi: 
            10.1016/j.jmb.2008.06.051. Epub 2008 Jun 24.
  PUBMED    18602401
REFERENCE   2  (bases 1 to 6569)
  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: 
            "10.1016/j.jmb.2008.06.051"; journalName: "J Mol Biol"; date: 
            "2008-09-19- 19"; volume: "381"; issue: "5"; pages: "1238-52"
COMMENT     Sequence Label: pCT-L7.5.1
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(9..27)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    68..571
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        608..712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(713..781,782..1573)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(931..950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1744..2332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2486..2503
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2656..2661,2662..2679,2680..2686)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2694..2710
                     /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     2699..2721
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2718..2734
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2718..2734
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2755..2773
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2938..3602
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3154..3271
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3566..3589
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3921..3932
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3933..3959
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4326..4355
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(4688..4707)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4689..4707)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4714..4731)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4714..4730)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4723..4745)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4875..5330
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4962..4981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5172..5193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5427..6101)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(6102..6383)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(6400..6419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6519..6541
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
       61 ttagacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg atggaataat
      121 ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt tagaaagtaa
      181 ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat
      241 ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga
      301 tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc
      361 tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa
      421 aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat
      481 tttttcttta atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat
      541 ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg
      601 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      661 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      721 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      781 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      841 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      901 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
      961 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1021 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1081 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1141 gaaggagcta accgcttttt ttcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1201 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1261 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1321 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1381 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1441 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1501 cagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1561 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1621 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1681 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1741 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1801 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1861 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1921 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1981 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2041 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2101 gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga
     2161 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2221 ggagcttcca ggggggaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2281 acttgagcgt cgatttttgt gatgctcgtc aggggggccg agcctatgga aaaacgccag
     2341 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2401 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2461 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     2521 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2581 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca
     2641 ttaggcaccc caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag
     2701 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tcggaattaa
     2761 ccctcactaa agggaacaaa agctgggtac ccgacaggtt atcagcaaca acacagtcat
     2821 atccattctc aattagctct accacagtgt gtgaaccaat gtatccagca ccacctgtaa
     2881 ccaaaacaat tttagaagta ctttcacttt gtaactgagc tgtcatttat attgaatttt
     2941 caaaaattct tacttttttt ttggatggac gcaaagaagt ttaataatca tattacatgg
     3001 cattaccacc atatacatat ccatatacat atccatatct aatcttactt atatgttgtg
     3061 gaaatgtaaa gagccccatt atcttagcct aaaaaaacct tctctttgga actttcagta
     3121 atacgcttaa ctgctcattg ctatattgaa gtacggatta gaagccgccg agcgggtgac
     3181 agccctccga aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa
     3241 acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt
     3301 tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga
     3361 atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta
     3421 attaatcagc gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca
     3481 taaccacttt aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta
     3541 ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa
     3601 accccggatc gaattcccta cttcatacat tttcaattaa gatgcagtta cttcgctgtt
     3661 tttcaatatt ttctgttatt gcttcagttt tagcacagga actgacaact atatgcgagc
     3721 aaatcccctc accaacttta gaatcgacgc cgtactcttt gtcaacgact actattttgg
     3781 ccaacgggaa ggcaatgcaa ggagtttttg aatattacaa atcagtaacg tttgtcagta
     3841 attgcggttc tcacccctca acaactagca aaggcagccc cataaacaca cagtatgttt
     3901 ttaaggacaa tagctcgacg attgaaggta gatacccata cgacgttcca gactacgctc
     3961 tgcaggctag tggtggtggt ggttctggtg gtggtggttc tggtggtggt ggttctgcta
     4021 gcgtttctga tgttccgagg gacctggaag ttgttgctgc gacctccacc agcctactga
     4081 tcagttggcg cggctacccc tgggctacct attatgggat catttacgga gaaacgggag
     4141 gaaatagcct tgtccaggag ttcactatgc ctggggttac caatgctacc atcagcggcc
     4201 ttaaacctgg agttgattat accatcactg tgtatgctgt cactcgcgtg gggcggacgt
     4261 ttgacacgcc gggcccaatc tccattaatt accgaacaga aattgacaaa ccatcccagg
     4321 gatccgaaca aaagcttatt tctgaagagg acttgtaata gctcgagatc tgataacaac
     4381 agtgtagatg taacaaaatc gactttgttc ccactgtact tttagctcgt acaaaataca
     4441 atatactttt catttctccg taaacaacat gttttcccat gtaatatcct tttctatttt
     4501 tcgttccgtt accaacttta cacatacttt atatagctat tcacttctat acactaaaaa
     4561 actaagacaa ttttaatttt gctgcctgcc atatttcaat ttgttataaa ttcctataat
     4621 ttatcctatt agtagctaaa aaaagatgaa tgtgaatcga atcctaagag aattgagctc
     4681 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     4741 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ccttcgccag
     4801 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4861 tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4921 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4981 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5041 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5101 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5161 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5221 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5281 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg
     5341 tattttctcc ttacgcatct gtgcggtatt tcacaccgca ggcaagtgca caaacaatac
     5401 ttaaataaat actactcagt aataacctat ttcttagcat ttttgacgaa atttgctatt
     5461 ttgttagagt cttttacacc atttgtctcc acacctccgc ttacatcaac accaataacg
     5521 ccatttaatc taagcgcatc accaacattt tctggcgtca gtccaccagc taacataaaa
     5581 tgtaagcttt cggggctctc ttgccttcca acccagtcag aaatcgagtt ccaatccaaa
     5641 agttcacctg tcccacctgc ttctgaatca aacaagggaa taaacgaatg aggtttctgt
     5701 gaagctgcac tgagtagtat gttgcagtct tttggaaata cgagtctttt aataactggc
     5761 aaaccgagga actcttggta ttcttgccac gactcatctc catgcagttg gacgatatca
     5821 atgccgtaat cattgaccag agccaaaaca tcctccttag gttgattacg aaacacgcca
     5881 accaagtatt tcggagtgcc tgaactattt ttatatgctt ttacaagact tgaaattttc
     5941 cttgcaataa ccgggtcaat tgttctcttt ctattgggca cacatataat acccagcaag
     6001 tcagcatcgg aatctagagc acattctgcg gcctctgtgc tctgcaagcc gcaaactttc
     6061 accaatggac cagaactacc tgtgaaatta ataacagaca tactccaagc tgcctttgtg
     6121 tgcttaatca cgtatactca cgtgctcaat agtcaccaat gccctccctc ttggccctct
     6181 ccttttcttt tttcgaccga attaattctt aatcggcaaa aaaagaaaag ctccggatca
     6241 agattgtacg taaggtgaca agctattttt caataaagaa tatcttccac tactgccatc
     6301 tggcgtcata actgcaaagt acacatatat tacgatgctg ttctattaaa tgcttcctat
     6361 attatatata tagtaatgtc gtttatggtg cactctcagt acaatctgct ctgatgccgc
     6421 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     6481 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     6541 gttttcaccg tcatcaccga aacgcgcga
//
