LOCUS       sequence-464150-        6095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of macrocypin-1a in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6095)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6095)
  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_3H6Q
FEATURES             Location/Qualifiers
     source          1..6095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(83..898)
                     /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(83..898)
                     /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     806..825
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     890..909
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1020..1608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1509..1528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1762..1779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1794..1933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1859..1878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2019..2041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2035..2226)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3035..4117)
                     /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     4079..4098
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4118..4195)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4401..4420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4508..4527
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4508..4526
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4527..4551
                     /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             4566..4588
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4611..4640
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5463..5480
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5533..5551)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5547..5594
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5631..6086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5718..5737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5928..5949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
       61 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
      121 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
      181 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
      241 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
      301 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
      361 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
      421 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
      481 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
      541 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
      601 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
      661 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
      721 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
      781 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
      841 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
      901 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
      961 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1021 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1081 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1141 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1201 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1261 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1321 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1681 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     1741 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     1801 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     1861 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     1921 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1981 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2041 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2101 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2161 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2221 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2281 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2341 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2401 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2461 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     2521 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     2581 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     2641 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     2701 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     2761 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     2821 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     2881 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     2941 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3001 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3061 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3121 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3181 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3241 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3301 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3361 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3421 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3481 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     3541 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     3601 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     3661 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     3721 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     3781 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     3841 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     3901 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     3961 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4021 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4081 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4141 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4201 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4261 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4321 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4381 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4441 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     4501 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     4561 ataattttgt ttaactttaa gaaggagata tacatatgat ggcgaagatt catcaccatc
     4621 atcaccatca ccatcaccac ggtagcctgc aagattcgga agtcaaccaa gaagcaaagc
     4681 cggaagtcaa gccggaagtg aaaccggaaa cccatattaa cctgaaagtt agtgacggca
     4741 gctctgaaat tttctttaag atcaaaaaga ccacgccgct gcgtcgcctg atggaagcgt
     4801 ttgccaaacg tcagggcaag gaaatggata gcctgcgttt cctgtatgac ggtattcgca
     4861 tccaggcaga tcaagcgccg gaagacctgg acatggaaga caacgacatc attgaagccc
     4921 accgtgaaca gattggcggc tctggaggat ttgaagatgg gttctataca atactacact
     4981 tggctgaggg ccagcatccg aactctaaga tcccgggcgg tatgtatgca agcagcaagg
     5041 acggcaaaga cgtgccggtt accgcggagc cgctgggtcc gcaaagcaag atccgttggt
     5101 ggattgcgcg tgatccgcaa gcgggtgatg atatgtacac cattaccgaa tttcgcatcg
     5161 ataacagcat tccgggtcag tggtcccgct ccccggtgga aaccgaagtt ccagtatacc
     5221 tgtatgatcg catcaaagcc gaggagactg gttatacgtg cgcttggcgt atccagcctg
     5281 cagaccatgg tgcggacggc gtttaccaca ttgttggtaa tgtgcgtata ggctcgaccg
     5341 actgggcgga cctcagagaa gagtatggtg aaccgcaagt ttacatgaaa ccggtcccag
     5401 tgattccgaa cgtctacatc ccgcgttggt tcatcctggg ctacggttcg taatgactcg
     5461 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     5521 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     5581 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     5641 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5701 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5761 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5821 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5881 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5941 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     6001 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     6061 taacaaaata ttaacgctta caatttaggt ggcac
//
