LOCUS       sequence-464133-        5936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of sprouty-related, EVH1 
            domain-containing protein 1 (Spred-1) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5936)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5936)
  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"
COMMENT     Sequence Label: pAIP4_1TJ6
FEATURES             Location/Qualifiers
     source          1..5936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     164..185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(414..1229)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(414..1229)
                     /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
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1137..1156
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1221..1240
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1351..1939
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1840..1859
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2093..2110
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2125..2264
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2190..2209)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2350..2372
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2366..2557)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3366..4448)
                     /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     4410..4429
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4449..4526)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4732..4751
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4839..4858
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4839..4857
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4858..4882
                     /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)."
     RBS             4897..4919
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4942..4971
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5635..5652
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5705..5723)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5719..5766
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5803..5936,1..322)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5890..5909)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
       61 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
      121 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      181 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
      241 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
      301 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      361 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
      421 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
      481 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
      541 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
      601 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
      661 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
      721 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
      781 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
      841 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
      901 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
      961 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     1021 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     1081 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     1141 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     1201 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     1261 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1321 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1381 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1441 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     1501 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1561 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1621 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1681 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1741 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1801 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1861 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1921 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1981 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2041 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2101 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2161 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2221 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2281 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2341 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2401 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     2461 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     2521 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     2581 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     2641 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     2701 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     2761 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     2821 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     2881 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     2941 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3001 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     3061 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     3121 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     3181 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     3241 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     3301 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     3361 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3421 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3481 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3541 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3601 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     3661 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     3721 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     3781 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     3841 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     3901 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     3961 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4021 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4081 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4141 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4201 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     4261 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     4321 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4381 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4441 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4501 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     4561 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     4621 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     4681 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     4741 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     4801 ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga
     4861 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     4921 atacatatga tggcgaagat tcatcaccat catcaccatc accatcacca cggtagcctg
     4981 caagattcgg aagtcaacca agaagcaaag ccggaagtca agccggaagt gaaaccggaa
     5041 acccatatta acctgaaagt tagtgacggc agctctgaaa ttttctttaa gatcaaaaag
     5101 accacgccgc tgcgtcgcct gatggaagcg tttgccaaac gtcagggcaa ggaaatggat
     5161 agcctgcgtt tcctgtatga cggtattcgc atccaggcag atcaagcgcc ggaagacctg
     5221 gacatggaag acaacgacat cattgaagcc caccgtgaac agattggcgg ctctggagat
     5281 tcatatgcaa gagtaagggc tgtcgttatg acccgtgacg actcgtccgg tggttggctg
     5341 caactgggcg gcggcggtct ttccagcgtt accgtcagca aaaccctgca gccgggtgac
     5401 agcggtggca ccgagttcct ggtgcacggc gaacgtctgc gcgacaagac ggtgatcctg
     5461 gagtgcgttc tgcgccgcga tctcgtttac aacaaggtga ctccgacgtt ccaccattgg
     5521 cgtatcggcg acaagaaatt tggtttgacc ttccagagcc cggcagatgc tcgtgcgttt
     5581 gatcgtggta ttagacgtgc gattgaagat ttgtctggtt cgtaatgact cgagcaccac
     5641 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     5701 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     5761 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     5821 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5881 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggcttt
//
