LOCUS       sequence-479224-        6898 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  ZBneg PANCS-binder negative AP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  AUTHORS   Styles MJ, Pixley JA, Wei T, Basile C, Lu SS, Dickinson BC
  TITLE     High-throughput protein binder discovery by rapid in vivo selection.
  JOURNAL   bioRxiv [Preprint]. 2025 Jan 17:2025.01.06.631531. doi: 
            10.1101/2025.01.06.631531.
  PUBMED    39829796
REFERENCE   2  (bases 1 to 6898)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Jan 17:2025.01.06.631531. doi: 
            10.1101/2025.01.06.631531."
COMMENT     Sequence Label: 73-79 ZBneg PANCS-binder negAP
FEATURES             Location/Qualifiers
     source          1..6898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(313..332)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             324..1139
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGLPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(397..416)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(1254..1271)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1424..1510
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1602..1629
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1671..1690
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1671..1689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1791..1808
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5894..6085
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6079..6101)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6187..6326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6242..6261
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6341..6358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6512..6898,1..202))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6592..6611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
       61 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      121 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
      181 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
      241 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
      301 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
      361 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
      421 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
      481 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
      541 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
      601 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
      661 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
      721 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
      781 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
      841 tgcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
      901 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
      961 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     1021 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     1081 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     1141 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1201 ccgcgcaaga ggacatccgg tggctgtttt ggcggatgag agaagatttt cagcctgata
     1261 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     1321 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt
     1381 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     1441 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     1501 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     1561 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     1621 tggccttttt gcgtttctac aaactcgctg aggagactta gggaccctac taatacgact
     1681 cactataggg agaaagacct gcaggtgcag taaggaaaaa aaaaatgaaa aaattattat
     1741 tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt catcaccatc
     1801 accatcacgc tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa aattcattta
     1861 ctaacgtctg gaaagacgac aaaactttag atcgttacgc taactatgag ggctgtctgt
     1921 ggaatgctac aggcgttgta gtttgtactg gtgacgaaac tcagtgttac ggtacatggg
     1981 ttcctattgg gcttgctatc cctgaaaatg agggtggtgg ctctgagggt ggcggttctg
     2041 agggtggcgg ttctgagggt ggcggtacta aacctcctga gtacggtgat acacctattc
     2101 cgggctatac ttatatcaac cctctcgacg gcacttatcc gcctggtact gagcaaaacc
     2161 ccgctaatcc taatccttct cttgaggagt ctcagcctct taatactttc atgtttcaga
     2221 ataataggtt ccgaaatagg cagggggcat taactgttta tacgggcact gttactcaag
     2281 gcactgaccc cgttaaaact tattaccagt acactcctgt atcatcaaaa gccatgtatg
     2341 acgcttactg gaacggtaaa ttcagagact gcgctttcca ttctggcttt aatgaggatc
     2401 cattcgtttg tgaatatcaa ggccaatcgt ctgacctgcc tcaacctcct gtcaatgctg
     2461 gcggcggctc tggtggtggt tctggtggcg gctctgaggg tggtggctct gagggtggcg
     2521 gttctgaggg tggcggctct gagggaggcg gttccggtgg tggctcttcc caaatggctc
     2581 aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat ttaccttccc
     2641 tccctcaatc ggttgaatgt cgcccttttg tctttggcgc tggtaaacct tacgagttca
     2701 gtatcgactg cgataagatc aacctgttcc gcggtgtctt tgcgtttctt ttatatgttg
     2761 ccacctttat gtatgtattt tctacgtttg ctaacatact gcgtaataag gagtcttaat
     2821 actagagtat ctgttagttt ttttctacta gagtatctgt tagttttttt ccatcgtggc
     2881 cggagtactt aagaagataa ctaaacaact aacggacaat tctacctagg atccctcgaa
     2941 aggaaaaaaa aaatggcaag cgagcagctg gaaaaggaat tacaagccct ggagaaagaa
     3001 cttgctcagc tgaagtggaa aaaccaagca ttggaaaaaa aactcgcgca gacgtcgggt
     3061 ggaagcggtg gttcagggtc ctcaggttcg ggtggaacta gtggctctgg tgggtcgggg
     3121 acaggcacag ggaccggaag tagcggatcg ggtggcacaa gtgggtctgg cggctctgga
     3181 tcaggcaccg gcagtgggtc ttcggggagt ggaggtacct caggctcagg aaaagcattt
     3241 atgcaagttg tcgaggctga catgctctct aagggtctac tcggtggcga ggcgtggtct
     3301 tcgtggcata aggaagactc tattcatgta ggagtacgct gcatcgagat gctcattgag
     3361 tcaaccggaa tggttagctt acaccgccaa aatgctggcg tagtaggtca agactctgag
     3421 actatcgaac tcgcacctga atacgctgag gctatcgcaa cccgtgcagg tgcgctggct
     3481 ggcatctctc cgatgttcca accttgcgta gttcctccta agccgtggac tggcattact
     3541 ggtggtggct attgggctaa cggtcgtcgt cctctggcgc tggtgcgtac tcacagtaag
     3601 aaagcactga tgcgctacga agacgtttac atgcctgagg tgtacaaagc gattaacatt
     3661 gcgcaaaaca ccgcatggaa aatcaacaag aaagtcctag cggtcgccaa cgtaatcacc
     3721 aagtggaagc attgtccggt cgaggacatc cctgcgattg agcgtgaaga actcccgatg
     3781 aaaccggaag acatcgacat gaatcctgag gctctcaccg cgtggaaacg tgctgccgct
     3841 gctgtgtacc gcaaggacaa ggctcgcaag tctcgccgta tcagccttga gttcatgctt
     3901 gagcaagcca ataagtttgc taaccataag gccatctggt tcccttacaa catggactgg
     3961 cgcggtcgtg tttacgctgt gtcaatgttc aacccgcaag gtaacgatat gaccaaagga
     4021 ctgcttacgc tggcgaaagg taaaccaatc ggtaaggaag gttactactg gctgaaaatc
     4081 cacggtgcaa actgtgcggg tgtcgataag gttccgttcc ctgagcgcat caagttcatt
     4141 gaggaaaacc acgagaacat catggcttgc gctaagtctc cactggagaa cacttggtgg
     4201 gctgagcaag attctccgtt ctgcttcctt gcgttctgct ttgagtacgc tggggtacag
     4261 caccacggcc tgagctataa ctgctccctt ccgctggcgt ttgacgggtc ttgctctggc
     4321 atccagcact tctccgcgat gctccgagat gaggtaggtg gtcgcgcggt taacttgctt
     4381 cctagtgaaa ccgttcagga catctacggg attgttgcta agaaagtcaa cgagattcta
     4441 caagcagacg caatcaatgg gaccgataac gaagtagtta ccgtgaccga tgagaacact
     4501 ggtgaaatct ctgagaaagt caagctgggc actaaggcac tggctggtca atggctggct
     4561 tacggtgtta ctcgcagtgt gactaagcgt tcagtcatga cgctggctta cgggtccaaa
     4621 gagttcggct tccgtcaaca agtgctggaa gataccattc agccagctat tgattccggc
     4681 aagggtctga tgttcactca gccgaatcag gctgctggat acatggctaa gctgatttgg
     4741 gaatctgtga gcgtgacggt ggtagctgcg gttgaagcaa tgaactggct taagtctgct
     4801 gctaagctgc tggctgctga ggtcaaagat aagaagactg gagagattct tcgcaagcgt
     4861 tgcgctgtgc attgggtaac tcctgatggt ttccctgtgt ggcaggaata caagaagcct
     4921 attcagacgc gcttgaacct gatgttcctc ggtcagttcc gcttacagcc taccattaac
     4981 accaacaaag atagcgagat tgatgcacac aaacaggagt ctggtatcgc tcctaacttt
     5041 gtacacagcc aagacggtag ccaccttcgt aagactgtag tgtgggcaca cgagaagtac
     5101 ggaatcgaat cttttgcact gattcacgac tccttcggta ccattccggc tgacgctgcg
     5161 aacctgttca aagcagtgcg cgaaactatg gttgacacat atgagtcttg tgatgtactg
     5221 gctgatttct acgaccagtt cgctgaccag ttgcacgagt ctcaattgga caaaatgcca
     5281 gcacttccgg ctaaaggtaa cttgaacctc cgtgacatct tagagtcgga cttcgcgttc
     5341 gcgtaaactt aattaacggc actcctcagc aaatataatg accctcttga taacccaaga
     5401 gggcattttt taatgcccat ggcgtccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     5461 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     5521 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     5581 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     5641 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     5701 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     5761 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     5821 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     5881 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     5941 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     6001 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     6061 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     6121 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     6181 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     6241 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     6301 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     6361 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6421 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6481 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6541 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6601 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6661 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6721 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6781 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6841 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgt
//
